Expression in filamentous fungi of protease inhibitors and variants thereof

ABSTRACT

Described herein are protease inhibitors, variants thereof and methods for their production.

FIELD OF THE INVENTION

This invention relates to methods for the expression of proteaseinhibitors and variants thereof in filamentous fungi. The inventiondiscloses fusion nucleic acids, vectors, fusion polypeptides, andprocesses for obtaining the protease inhibitors.

BACKGROUND OF THE INVENTION

Proteases are involved in a wide variety of biological processes.Disruption of the balance between proteases and protease inhibitors isoften associated with pathologic tissue destruction.

Various studies have focused on the role of proteinases in tissueinjury, and it is thought that the balance between proteinases andproteinase inhibitors is a major determinant in maintaining tissueintegrity. Serine proteinases from inflammatory cells, includingneutrophils, are implicated in various inflammatory disorders, such aspulmonary emphysema, arthritis, atopic dermatitis and psoriasis.

Proteases also appear to function in the spread of certain cancers.Normal cells exist in contact with a complex protein network, called theextracellular matrix (ECM). The ECM is a barrier to cell movement andcancer cells must devise ways to break their attachments, degrade, andmove through the ECM in order to metastasize. Proteases are enzymes thatdegrade other proteins and have long been thought to aid in freeing thetumor cells from their original location by chewing up the ECM. Recentstudies have suggested that they may promote cell shape changes andmotility through the activation of a protein in the tumor cell membranecalled Protease-Activated Receptor-2 (PAR2). This leads to a cascade ofintracellular reactions that activates the motility apparatus of thecell. Thus, it is hypothesized that one of the first steps in tumormetastasis is a reorganization of the cell shape such that it forms adistinct protrusion at one edge facing the direction of migration. Thecell then migrates through a blood vessel wall and travels to distallocations, eventually reattaching and forming a metastatic tumor. Forexample, human prostatic epithelial cells constitutively secreteprostate-specific antigen (PSA), a kallikrein-like serine protease,which is a normal component of the seminal plasma. The protease acts todegrade the extracellular matrix and facilitate invasion of cancerouscells.

Synthetic and natural protease inhibitors have been shown to inhibittumor promotion in vivo and in vitro. Previous research investigationshave indicated that certain protease inhibitors belonging to a family ofstructurally-related proteins classified as serine protease inhibitorsor SERPINS, are known to inhibit several proteases including trypsin,cathepsin G, thrombin, tissue kallikrein, as well as neutrophilelastase. The SERPINS are extremely effective at preventing/suppressingcarcinogen-induced transformation in vitro and carcinogenesis in animalmodel systems. Systemic delivery of purified protease inhibitors reducesjoint inflammation and cartilage and bone destruction as well.

Topical administration of protease inhibitors finds use in suchconditions as atopic dermatitis, a common form of inflammation of theskin, which may be localized to a few patches or involve large portionsof the body. The depigmenting activity of protease inhibitors and theircapability to prevent ultraviolet-induced pigmentation have beendemonstrated both in vitro and in vivo. Paine et al., Journal ofInvestigative Dermatology 116, 587-595 (2001). Also, protease inhibitorshave been found to help wound healing(http://www.sciencedaily.com/releases/2000/10/001002071718.htm).Secretory leukocyte protease inhibitor was demonstrated to reverse thetissue destruction and speed the wound healing process when appliedtopically. In addition, serine protease inhibitors can also help toreduce pain in lupus erythematosus patients (See U.S. Pat. No.6,537,968).

As noted above, protease inhibitors interfere with the action ofproteases. Naturally occurring protease inhibitors can be found in avariety of foods such as cereal grains (oats, barley, and maize),Brussels sprouts, onion, beetroot, wheat, finger millet, and peanuts.One source of interest is the soybean. The average level in soybeans isaround 1.4 percent and 0.6 percent for Kunitz and Bowman-Birkrespectively, two of the most important protease inhibitors. These lowlevels make it impractical to isolate the natural protease inhibitor forclinical applications.

Thus, there is a need for a method to produce large quantities ofprotease inhibitors and their variants that also reduces or eliminatesthe risk associated with blood-borne infectious agents when these agentsare produced in mammalian tissue culture cells. The inventive productionmethod provided for herein allows for the manufacture of largequantities of the protein therapeutic.

BRIEF SUMMARY OF THE INVENTION

Provided herein are nucleic acids, cells and methods for the productionof protease inhibitors and variants thereof.

In a first embodiment, nucleic acids encoding a functional proteaseinhibitor are provided. In one aspect, a nucleic acid comprisingregulatory sequences operatively linked to a first, second, third andfourth nucleic acid sequences are provided. Terminator sequences areprovided following the fourth nucleic acid sequence.

In a second aspect, the first nucleic acid sequence encodes a signalpolypeptide functional as a secretory sequence in a first filamentousfungus, the second nucleic acid encodes a secreted polypeptide orfunctional portion thereof normally secreted from said first or a secondfilamentous fungus, the third nucleic acid encodes a cleavable linkerand the fourth nucleic acid encodes a protease inhibitor or fragmentthereof.

In a third aspect, an expression cassette comprising nucleic acidsequences encoding a protease inhibitor is provided.

In fourth aspect the present invention relates to a polynucleotideencoding a protease inhibitor variant. The polynucleotide may encode aBowman-Birk Inhibitor variant wherein at least one loop has beenaltered. The polynucleotide may encode a Soybean Trypsin Inhibitorvariant wherein at least one loop has been altered.

In a second embodiment, methods of expressing a functional proteaseinhibitor or variant thereof are provided. In one aspect, a host cell is(i) transformed with an expression cassette comprising a nucleic acidsequence encoding a protease inhibitor or variant thereof, and (ii)cultured under appropriate conditions to express the protease inhibitoror variants thereof. Optionally, the method further comprises recoveringthe protease inhibitor or variant thereof.

In a second aspect, a host cell is (i) transformed with an firstexpression cassette comprising a nucleic acid sequence encoding aprotease inhibitor or variant thereof, (ii) transformed with a secondexpression cassette comprising a nucleic acid sequence encoding achaperone, and (iii) cultured under appropriate conditions to expressthe protease inhibitors or variant thereof. Optionally, the proteaseinhibitors or variant thereof may be recovered. In one aspect, theprotease inhibitors or variant thereof are expressed as a fusionprotein. Optionally, the method further comprises recovering theprotease inhibitor or variant thereof.

In a third embodiment, cells capable of expressing a protease inhibitoror variant thereof is provided. Host cells are transformed an expressioncassette encoding a protease inhibitor or variant thereof. Host cellsmay be selected from the group consisting of Aspergillus andTrichoderma.

In a fourth embodiment, a functional protease inhibitor or variantthereof is provided. In one aspect, the functional protease inhibitor orvariant thereof is expressed as a fusion protein consisting of theglucoamylase signal sequence, prosequence, catalytic domain and linkerregion up to amino acid number 502 of mature glucoamylase, followed byamino acids NVISKR and then by the mature protease inhibitor or variantthereof.

In a second aspect, the expressed proteins are treated with a proteaseto liberate a protease inhibitor or variant thereof from the fusionprotein.

In a third aspect, the present invention provides a polypeptide havingprotease inhibitory activity, selected from the group consisting of

-   -   a) Bowman-Birk Inhibitor variants;    -   b) Soybean Trypsin Inhibitor variants;    -   c) Bowman-Birk Inhibitor;    -   d) Soybean Trypsin Inhibitor; and    -   e) A scaffold comprising at least one variant sequence.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the scope and spirit of the invention will becomeapparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the codon optimized nucleotide sequence for soybeanBowman-Birk type protease inhibitor (BBI) (SEQ ID NO:1). This sequenceincludes nucleotides encoding NVISKR (dotted underline), the cleavagesite for the fusion protein and three restriction enzyme sites forcloning into the expression plasmid. The NheI site at the 5′ end andXhoI site at the 3′ end are underlined and labeled. The BstEII site atthe 3′ end is designated by the # symbols. The stop codon is designatedby the asterisks. There is no start codon as this is expressed as afusion protein. The mature BBI coding sequence is indicated by thedouble underline (SEQ ID NO:2). The addition of nucleotides encodingthree glycine (FIG. 1B) residues prior to the mature BBI coding sequencecan be done using the sequence encoding the three glycine residuesindicated in FIG. 2 (SEQ ID NO:5). FIG. 1C nucleotide sequence encodingBBI, the three restriction sites, the kex2 site, three glycine residuesat the N-terminal end and six histidine residues at the C-terminal endis shown (SEQ ID NO:54).

FIG. 2 is the codon optimized nucleotide sequence for Soybean TrypsinInhibitor (STI), a Kunitz type protease inhibitor (SEQ ID NO:3). Thissequence includes nucleotides encoding NVISKR (dotted underline) (SEQ IDNO:4), the cleavage site for the fusion protein, and six histidineresidues at the C-terminal end (indicated by the dots). Threerestriction enzyme sites (NheI at 5′ end and XhoI and BstEII at 3′ end,indicated as described for FIG. 1) for cloning into the expressionplasmid were also included. The three glycine residues after the kex2site (NVISKR) are indicated by bold. The nucleotide sequence encodingthe mature STI is indicated by the dashed underline (SEQ 1N NO:6).

FIG. 3A is the mature amino acid sequence for BBI (SEQ ID NO:7). FIG. 3Bis BBI with three glycine residues at N-terminal (SEQ ID NO:8). FIG. 3Cis BBI with three glycine residues at N-terminal end and six histidineresidues at C-terminal end (SEQ ID NO:9). In FIGS. 3A-C Loop1 isindicated by the underlined amino acid residues and Loop II amino acidresidues are indicated by the bold type.

FIG. 4A is the mature STI with three glycine residues at the N-terminusand with six histidine residues at the C-terminus (SEQ ID NO: 10). FIG.4B is STI with three glycine residues at the N-terminal end (SEQ IDNO:11). FIG. 4C is the mature amino acid sequence for STI (SEQ IDNO:12). Loop1 is indicated by the underlined amino acid residues (SEQ IDNO:13). Loop II amino acid residues are indicated by the bold type (SEQID NO:14).

FIG. 5 is a diagram of the expression plasmid pSLGAMpR2-BBI. Thisplasmid is based on pSLGAMpR2 which is derived from pSL1180 by insertingthe A. niger glucoamylase promoter, catalytic core and terminator, amarker gene (A. niger pyrG) and a bovine prochymosin gene. The pSL 1180plasmid is available from Amersham Biosciences (Piscataway, N.J.). ThepSLGAMpR2 plasmid has the elements listed above inserted in the samerelative location as shown for pSLGAMpR2-BBI except that the bovineprochymosin gene is located where the BBI gene. Thus, the BBI genereplaces the prochymosin gene in pSLGAMpR2 to yield pSLGAMpR2-BBI.

FIG. 6 is the amino acid sequences for wild-type BBI (SEQ ID NO:7) andselect variants of BBI (SEQ ID NOs:15 thru 29). The wild-type BBI hasthe loops underlined. The differences in the variants from the wild-typeare shown as either bold/underlined (Loop I) or bold (LoopII). In somevariants, e.g., C2, C3, C4, C5 and Factor B, alanine at position13(between two cysteines) was also changed to either “Serine”, “Glycine”or “Glutamine”. Also, compstatin peptide has 9 amino acids instead of 7.The variant sequences are also shown (SEQ ID NOs:30 thru 40).

FIG. 7 is a photograph of a protein SDS gel. Lane 1 contains molecularweight markers. Lane 2 is the untransformed parental strain. Lane 3 isthe parental strain transformed with BBI-encoding DNA. Lane 4 is theparental strain co-transformed with a BBI-encoding vector and achaperone (pdiA)-encoding vector. Lane 15 is the parental strainco-transformed with a BBI-encoding vector and a chaperone(prpA)-encoding vector. Expression of the desired protein, e.g., BBI,was enhanced in the presence of the chaperone.

FIG. 8 is a diagram of the plasmid pTrex4.

FIG. 9A-D is the nucleic acid sequence for pTrex2 (SEQ ID NO:41).

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER C OLLINS DICTIONARYOF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively. Practitioners are particularly directed toSambrook et al., 1989, and Ausubel F M et al., 1993, for definitions andterms of the art. It is to be understood that this invention is notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

Definitions

An “expression cassette” or “expression vector” is a nucleic acidconstruct generated recombinantly or synthetically, with a series ofspecified nucleic acid elements that permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid sequence to betranscribed and a promoter. Expression cassette may be usedinterchangeably with DNA construct and its grammatical equivalents.

As used herein, the term “vector” refers to a nucleic acid constructdesigned to transfer nucleic acid sequences into cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in some eukaryotes orintegrates into the host chromosomes.

The term “nucleic acid molecule” or “nucleic acid sequence” includesRNA, DNA and cDNA molecules. It will be understood that, as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding a given protein may be produced.

As used herein, a “fusion DNA sequence” comprises from 5′ to 3′ a first,second, third and fourth DNA sequences.

As used herein, “a first nucleic acid sequence” or “first DNA sequence”encodes a signal peptide functional as a secretory sequence in a firstfilamentous fungus. Such signal sequences include those fromglucoamylase, α-amylase and aspartyl proteases from Aspergillus nigervar. awamori, Aspergillus niger, Aspergillus oryzae, signal sequencesfrom cellobiohydrolase I, cellobiohydrolase II, endoglucanase I,endoglucanase III from Trichoderma, signal sequences from glucoamylasefrom Neurospora and Humicola as well as signal sequences from eukaryotesincluding the signal sequence from bovine chymosin, human issueplasminogen activator, human interferon and synthetic consensuseukaryotic signal sequences such as that described by Gwynne et al.(1987) Bio/Technology 5, 713-719. Particularly preferred signalsequences are those derived from polypeptides secreted by the expressionhost used to express and secrete the fusion polypeptide. For example,the signal sequence from glucoamylase from Aspergillus niger ispreferred when expressing and secreting a fusion polypeptide fromAspergillus niger. As used herein, first amino acid sequences correspondto secretory sequences which are functional in a filamentous fungus.Such amino acid sequences are encoded by first DNA sequences as defined.

As used herein, “second DNA sequences” encode “secreted polypeptides”normally expressed from filamentous fungi. Such secreted polypeptidesinclude glucoamylase, α-amylase and aspartyl proteases from Aspergillusniger var. awamori, Aspergillus niger, and Aspergillus oryzae,cellobiohydrolase 1, cellobiohydrolase II, endoglucanase I andendoglucanase III from Trichoderma and glucoamylase from Neurosporaspecies and Humicola species. As with the first DNA sequences, preferredsecreted polypeptides are those which are naturally secreted by thefilamentous fungal expression host. Thus, for example when usingAspergillus niger, preferred secreted polypeptides are glucoamylase andα-amylase from Aspergillus niger, most preferably glucoamylase. In oneaspect the glucoamylase is greater than 95%, 96%, 97%, 98% or 99%homologous with an Aspergillus glucoamylase.

When Aspergillus glucoamylase is the secreted polypeptide encoded by thesecond DNA sequence, the whole protein or a portion thereof may be used,optionally including a prosequence. Thus, the cleavable linkerpolypeptide may be fused to glucoamylase at any amino acid residue fromposition 468-509. Other amino acid residues may be the fusion site bututilizing the above residues is particularly advantageous.

A “functional portion of a secreted polypeptide” or grammaticalequivalents means a truncated secreted polypeptide that retains itsability to fold into a normal, albeit truncated, configuration. Forexample, in the case of bovine chymosin production by A. niger var.awamori it has been shown that fusion of prochymosin following the 11thamino acid of mature glucoamylase provided no benefit compared toproduction of preprochymosin (U.S. Pat. No. 5,364,770). In U.S. Ser. No.08/318,494, it was shown that fusion of prochymosin onto the C-terminusof preproglucoamylase up to the 297th amino acid of mature glucoamylaseplus a repeat of amino acids 1-11 of mature glucoamylase yielded nosecreted chymosin in A. niger var. awamori. In the latter case it isunlikely that the portion (approximately 63%) of the glucoamylasecatalytic domain present in the fusion protein was able to foldcorrectly so that an aberrant, mis-folded and/or unstable fusion proteinmay have been produced which could not be secreted by the cell. Theinability of the partial catalytic domain to fold correctly may haveinterfered with the folding of the attached chymosin. Thus, it is likelythat sufficient residues of a domain of the naturally secretedpolypeptide must be present to allow it to fold in its normalconfiguration independently of the desired polypeptide to which it isattached.

In most cases, the portion of the secreted polypeptide will be bothcorrectly folded and result in increased secretion as compared to itsabsence.

Similarly, in most cases, the truncation of the secreted polypeptidemeans that the functional portion retains a biological function. In apreferred embodiment, the catalytic domain of a secreted polypeptide isused, although other functional domains may be used, for example, thesubstrate binding domains. In the case of Aspergillus niger andAspergillus niger var. awamori glucoamylase, preferred functionalportions retain the catalytic domain of the enzyme, and include aminoacids 1-471. Additionally preferred embodiments utilize the catalyticdomain and all or part of the linker region. Alternatively, the starchbinding domain of glucoamylase may be used, which comprises amino acids509-616 of Aspergillus niger and Aspergillus niger var. awamoriglucoamylase.

As used herein, “third DNA sequences” comprise DNA sequences encoding acleavable linker polypeptide. Such sequences include those which encodethe prosequence of glucoamylase, the prosequence of bovine chymosin, theprosequence of subtilisin, prosequences of retroviral proteasesincluding human immunodeficiency virus protease and DNA sequencesencoding amino acid sequences recognized and cleaved by trypsin, factorX_(a) collagenase, clostripin, subtilisin, chymosin, yeast KEX2protease, Aspergillus KEXB and the like. See e.g. Marston, F. A. O.(1986) Biol. Chem J. 240, 1-12. Such third DNA sequences may also encodethe amino acid methionine that may be selectively cleaved by cyanogenbromide. It should be understood that the third DNA sequence need onlyencode that amino acid sequence which is necessary to be recognized by aparticular enzyme or chemical agent to bring about cleavage of thefusion polypeptide. Thus, the entire prosequence of, for example,glucoamylase, chymosin or subtilisin need not be used. Rather, only thatportion of the prosequence which is necessary for recognition andcleavage by the appropriate enzyme is required.

It should be understood that the third nucleic acid need only encodethat amino acid sequence which is necessary to be recognized by aparticular enzyme or chemical agent to bring about cleavage of thefusion polypeptide.

Particularly preferred cleavable linkers are the KEX2 proteaserecognition site (Lys-Arg), which can be cleaved by a native AspergillusKEX2-like (KEXB) protease, trypsin protease recognition sites of Lys andArg, and the cleavage recognition site for endoproteinase-Lys-C.

As used herein, “fourth DNA sequences” encode “desired polypeptides.”Such desired polypeptides include protease inhibitors and variantsthereof.

The above-defined four DNA sequences encoding the corresponding fouramino acid sequences are combined to form a “fusion DNA sequence.” Suchfusion DNA sequences are assembled in proper reading frame from the 5′terminus to 3′ terminus in the order of first, second, third and fourthDNA sequences. As so assembled, the DNA sequence will encode a “fusionpolypeptide” or “fusion protein” or “fusion analog” encoding from itsamino-terminus a signal peptide functional as a secretory sequence in afilamentous fungus, a secreted polypeptide or portion thereof normallysecreted from a filamentous fungus, a cleavable linker polypeptide and adesired polypeptide.

As used herein, the terms “desired protein” or “desired polypeptide”refers to a polypeptide or protein in its mature form that is not fusedto a secretion enhancing construct. Thus, a “desired protein” or“desired polypeptide” refers to the protein to be expressed and secretedby the host cell in a non-fused form.

As used herein, a “fusion polypeptide” or “fusion protein” or “fusionanalog” encodes from its amino-terminus a signal peptide functional as asecretory sequence functional in a host cell, a secreted polypeptide orportion thereof normally secreted from a host cell, a cleavable linkerpolypeptide and a desired polypeptide. The fusion protein may beprocessed by host cell enzymes, e.g., a protease, to yield the desiredprotein free from the other protein sequences in the fusion protein. Asused herein, the terms “fusion analog” or “fusion polypeptide” or“fusion protein” may be used interchangeably.

As used herein, a “promoter sequence” is a DNA sequence which isrecognized by the particular filamentous fungus for expression purposes.It is operably linked to a DNA sequence encoding the above definedfusion polypeptide. Such linkage comprises positioning of the promoterwith respect to the translation initiation codon of the DNA sequenceencoding the fusion DNA sequence. The promoter sequence containstranscription and translation control sequences which mediate theexpression of the fusion DNA sequence. Examples include the promoterfrom the A. niger var. awamori or A. niger glucoamylase genes (Nunberg,J. H. et al. (1984) Mol. Cell. Biol. 4, 2306-2315; Boel, E. et al.(1984) EMBO J. 3, 1581-1585), the A. oryzae, A. niger var. awamori or A.niger or alpha-amylase genes, the Rhizomucor miehei carboxyl proteasegene, the Trichoderma reesei cellobiohydrolase I gene (Shoemaker, S. P.et al. (1984) European Patent Application No. EPO0137280A1), the A.nidulans trp C gene (Yelton, M. et al. (1984) Proc. Natl. Acad. Sci. USA81, 1470-1474; Mullaney, E. J. et al. (1985) Mol. Gen. Genet. 199,37-45) the A. nidulans alcA gene (Lockington, R. A. et al. (1986) Gene33 137-149), the A. nidulans amdS gene (McKnight, G. L. et al. (1986)Cell 46, 143-147), the A. nidulans amdS gene (Hynes, M. J. et al. (1983)Mol. Cell Biol. 3, 1430-1439), and higher eukaryotic promoters such asthe SV40 early promoter (Barclay, S. L. and E. Meller (1983) Molecularand Cellular Biology 3, 2117-2130).

Likewise a “terminator sequence” is a DNA sequence which is recognizedby the expression host to terminate transcription. It is operably linkedto the 3′ end of the fusion DNA encoding the fusion polypeptide to beexpressed. Examples include the terminator from the A. nidulans trpCgene (Yelton, M. et al. (1984) Proc. Natl. Acad. Sci. USA 81, 1470-1474;Mullaney, E. J. et al. (1985) Mol. Gen. Genet. 199, 37-45), the A. nigervar. awamori or A. niger glucoamylase genes (Nunberg, J. H. et al.(1984) Mol. Cell. Biol. 4, 2306-253; Boel, E. et al. (1984) EMBO J. 3,1581-1585), the A. oryzae, A. niger var. awamori or A. niger oralpha-amylase genes and the Rhizomucor miehei carboxyl protease gene(EPO Publication No. 0 215 594), although any fungal terminator islikely to be functional in the present invention.

A “polyadenylation sequence” is a DNA sequence which when transcribed isrecognized by the expression host to add polyadenosine residues totranscribed mRNA. It is operably linked to the 3′ end of the fusion DNAencoding the fusion polypeptide to be expressed. Examples includepolyadenylation sequences from the A. nidulans trpC gene (Yelton, M. etal. (1984) Proc. Natl. Acad. Sci. USA 81, 1470-1474; Mullaney, E. J. etal. (1985) Mol. Gen. Genet. 199, 37-45), the A. niger var. awamori or A.niger glucoamylase genes (Nunberg, J. H. et al. (1984) Mol. Cell. Biol.4, 2306-2315) (Boel, E. et al. (1984) EMBO J. 3, 1581-1585), the A.oryzae, A. niger var. awamori or A. niger or alpha-amylase genes and theRhizomucor miehei carboxyl protease gene described above. Any fungalpolyadenylation sequence, however, is likely to be functional in thepresent invention.

As used herein, the term “selectable marker-encoding nucleotidesequence” refers to a nucleotide sequence which is capable of expressionin fungal cells and where expression of the selectable marker confers tocells containing the expressed gene the ability to grow in the presenceof a corresponding selective condition.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNAencoding a secretory leader is operably linked to DNA for a polypeptideif it is expressed as a preprotein that participates in the secretion ofthe polypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence; or a ribosomebinding site is operably linked to a coding sequence if it is positionedso as to facilitate translation. Generally, “operably linked” means thatthe DNA sequences being linked are contiguous, and, in the case of asecretory leader, contiguous and in reading phase. However, enhancers donot have to be contiguous. Linking is accomplished by ligation atconvenient restriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation. It follows thatthe term “protease inhibitor expression” refers to transcription andtranslation of the specific protease inhibitors and variants thereofgene to be expressed, the products of which include precursor RNA, mRNA,polypeptide, post-translation processed polypeptide, and derivativesthereof. Similarly, “protease inhibitor expression” refers to thetranscription, translation and assembly of protease inhibitors andvariants thereof into a form exemplified by FIG. 6. By way of example,assays for protease inhibitor expression include examination of fungalcolonies when exposed to the appropriate conditions, western blot forprotease inhibitor protein, as well as northern blot analysis andreverse transcriptase polymerase chain reaction (RT-PCR) assays forprotease inhibitor mRNA.

As used herein the term “glycosylated” means that oligosaccharidemolecules have been added to particular amino acid residues on aprotein. A “de-glycosylated” protein is a protein that has been treatedto partially or completely remove the oligosaccharide molecules from theprotein. An “aglycosylated” protein is a protein that has not had theoligosaccharide molecules added to the protein. This may be due to amutation in the protein that prevents the addition of theoligosaccharide.

A “non-glycosylated” protein is a protein that does not have theoligosaccharide attached to the protein. This may be due to variousreasons, including but not limited to, the absence of enzymesresponsible for the addition of the oligosaccharides to proteins. Theterm “non-glycosylated” encompasses both proteins that have not had theoligosaccharide added to the protein and those in which theoligosaccharides have been added but were subsequently removed. An“aglycosylated” protein may be a “non-glycosylated” protein. A“non-glycosylated” protein may be either an “aglycosylated” protein or a“deglycosylated” protein.

The terms “isolated” or “purified” as used herein refer to a nucleicacid or polypeptide that is removed from at least one component withwhich it is naturally associated.

The term “substantially free” includes preparations of the desiredpolypeptide having less than about 20% (by dry weight) other proteins(i.e., contaminating protein), less than about 10% other proteins, lessthan about 5% other proteins, or less than about 1% other proteins.

The term “substantially pure” when applied to the proteins or fragmentsthereof of the present invention means that the proteins are essentiallyfree of other substances to an extent practical and appropriate fortheir intended use. In particular, the proteins are sufficiently pureand are sufficiently free from other biological constituents of the hostcells so as to be useful in, for example, protein sequencing, orproducing pharmaceutical preparations.

The term “target protein” as used herein refers to protein, e.g., anenzyme, hormone or the like, whose action would be blocked by thebinding of the variant inhibitors provided for herein.

The terms “variant sequence” or “variant sequences” refer to the shortpolypeptide sequence(s) that replace the binding loops of the wild-typeprotease inhibitor or other scaffold. The variant sequence does not needto be of the same length as the binding loop sequence it is replacing inthe scaffold.

The term “scaffold” refers to the wild-type protein sequence into whicha variant sequence may be introduced. In an embodiment the scaffold willhave portions, e.g., loops, that may be replaced. For example, the STIand BBI sequences used herein would be a scaffold for a variantsequence.

Protease Inhibitors

Two protein protease inhibitors have been isolated from soybeans, theKunitz-type trypsin inhibitor (soybean trypsin inhibitor, STI) and theBowman-Birk protease inhibitor (BBI). See, e.g., Birk, Int. J. Pept.Protein Res. 25: 113-131 (1985) and Kennedy, Am. J. Clin. Neutr. 68:1406S-1412S (1998). These inhibitors serve as a scaffold for the variantsequences.

In addition, to alterations in the scaffold comprising the variantsequences, other desired proteins used herein include the addition ofthree glycine residues at the N-terminal and/or six histidine residuesat the C-terminal. See FIGS. 3 and 4.

Soybean Trypsin Inhibitor (STI)

STI inhibits the proteolytic activity of trypsin by the formation of astable stoichiometric complex. See, e.g., Liu, K., Chemistry andNutritional value of soybean components. In: Soybeans, chemistry,technology and utilization. pp. 32-35 (Aspen publishers, Inc.,Gaithersburg, Md., 1999). STI consists of 181 amino acid residues withtwo disulfide bridges and is roughly spherically shaped. See, e.g., Songet al., J. Mol. Biol. 275: 347-63 (1998). The two disulfide bridges formtwo binding loops similar to those described below for BBI.

The Kunitz-type soybean trypsin inhibitor (STI) has played a key role inthe early study of proteinases, having been used as the main substratein the biochemical and kinetic work that led to the definition of thestandard mechanism of action of proteinase inhibitors.

Bowman-Birk Inhibitor (BBI)

BBI proteins are a kinetically and structurally well-characterizedfamily of small proteins (60-90 residues) isolated from leguminousseeds. They have a symmetrical structure of two tricyclic domains eachcontaining an independent binding loop. Loop I typically inhibitstrypsin and loop II chymotrypsin (Chen et al., J. Biol. Chem. (1992)267: 1990-1994; Werner & Wemmer, 1992; Lin et al., Eur. J. Biochem.(1993) 212: 549-555; Voss et al., Eur. J. Biochem. (1996) 242: 122-131).These binding regions each contain a “canonical loop” structure, whichis a motif found in a variety of serine proteinase inhibitors (Bode &Huber, Eur. J. Biochem. (1992) 204: 433-451).

BBI is an 8 k-Da protein that inhibits the proteases trypsin andchymotrypsin at separate reactive sites. See, e.g., Billings et al.,Pro. Natl. Acad. Sci. 89: 3120-3124 (1992). STI and BBI are found onlyin the soybean seed, and not in any other part of the plant. See, e.g.,Birk, Int. J. Pept. Protein Res. 25: 113-131 (1985).

Although numerous isoforms of BBI have been characterized, SEQ ID NO: 7(FIG. 3) shows the amino acid sequence of the BBI backbone used hereincomprising approximately 71 amino acid residues. In addition, BBI maybecome truncated with as many as 10 amino acid residues being removedfrom either the N- or C-terminal. For example, upon seed desiccation, aBBI may have the C-terminal 9 or 10 amino acid residues removed. Thus,proteolysis is highly tolerated prior to the initial disulphide and justafter the terminal disulphide bond, the consequences of which areusually not detrimental to the binding to target protein. However, itwill be appreciated that any one of the isoforms or truncated formscould be used.

Protease Inhibitor Variants

As noted above, the STI and BBI protease inhibitors have binding loopsthat inhibit proteases. The inventive protease inhibitor variantsprovided for herein have alterations in Loop I, Loop II or both loops.In an embodiment, the loops are replaced with sequences that interactwith a target protein.

The loops can be replaced with sequences derived from VEGF bindingproteins, inhibitors of the complement pathway such as C2, C3, C4 or C5inhibitors, cotton binding proteins, Compstatin and the like.Alternatively, variant sequences can be selected by various methodsknown in the art such as, for example, phage display or other screeningmethod. For example, a random peptide gene library is fused with phagePIII gene so the peptide library will be displayed on the surface of thephage. Subsequently, the phage display library is exposed to the targetprotein and washed with buffer to remove non-specific binding (thisprocess is sometimes referred to as panning). Finally, the binding phageand PCR the DNA sequence for the peptide encoded are isolated.

Generally, a loop will be replaced with a variant sequence, i.e.,peptides, 3 to 14 amino acids in length, 5 to 10 amino acids beingpreferred. Longer sequences may be used as long as they provide thebinding and/or inhibition desired. In addition, peptides suitable foruse as replacements of the binding loop(s) should adopt a functionalconformation when contained within a constrained loop, i.e., a loopformed by the presence to a disulfide bond between two cysteineresidues. In specific embodiments, the peptides are between 7 and 9amino acids in length. These replacement sequences also provide proteaseinhibition or binding to the targeted proteins.

In some cases it may be advantages to alter a single amino acid.Specifically, the Alanine at residue 13 of wild-type STI or BBI may bechanged to a Serine, a Glycine or a Glutamine.

Fusion Proteins

Each protease inhibitor and variant thereof will be expressed as afusion protein by the host fungal cell. Although cleavage of the fusionpolypeptide to release the desired protein will often be useful, it isnot necessary. Protease inhibitors and variants thereof expressed andsecreted as fusion proteins surprisingly retain their function.

The above-defined four DNA sequences encoding the corresponding fouramino acid sequences are combined to form a “fusion DNA sequence.” Suchfusion DNA sequences are assembled in proper reading frame from the 5′terminus to 3′ terminus in the order of first, second, third and fourthDNA sequences. As so assembled, the DNA sequence will encode a “fusionpolypeptide” encoding from its amino-terminus a signal peptidefunctional as a secretory sequence in a filamentous fungus, a secretedpolypeptide or portion thereof normally secreted from a filamentousfungus, a cleavable linker peptide and a desired polypeptide, e.g., aprotease inhibitor and variants thereof.

Production of fusion proteins can be accomplished by use of the methodsdisclosed in, for example, U.S. Pat. Nos. 5,411,873, 5,429,950, and5,679,543. Other methods are well known in the art.

Expression of Recombinant a Protease Inhibitor

To the extent that this invention depends on the production of fusionproteins, it relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Ausubel et al., eds., Current Protocols inMolecular Biology (1994).

This invention provides filamentous fungal host cells which have beentransduced, transformed or transfected with an expression vectorcomprising a protease inhibitor-encoding nucleic acid sequence. Theculture conditions, such as temperature, pH and the like, are thosepreviously used for the parental host cell prior to transduction,transformation or transfection and will be apparent to those skilled inthe art.

Basically, a nucleotide sequence encoding a fusion protein is operablylinked to a promoter sequence functional in the host cell. Thispromoter-gene unit is then typically cloned into intermediate vectorsbefore transformation into the host cells for replication and/orexpression. These intermediate vectors are typically prokaryoticvectors, e.g., plasmids, or shuttle vectors.

In one approach, a filamentous fungal cell line is transfected with anexpression vector having a promoter or biologically active promoterfragment or one or more (e.g., a series) of enhancers which functions inthe host cell line, operably linked to a nucleic acid sequence encodinga protease inhibitor, such that the a protease is expressed in the cellline. In a preferred embodiment, the DNA sequences encode a proteaseinhibitor or variant thereof. In another preferred embodiment, thepromoter is a regulatable one.

A. Codon Optimization

Optimizing codon usage in genes that express well with those genes thatdo not express well is known in the art. See Barnett et al., GB2200118and Bergquist et al., Extremophiles (2002) 6: 177-184. Codonoptimization, as used herein, was based on comparing heterologousproteins that are expressed well in Aspergillus and native secretedproteins to the heterologous proteins that are not expressed well. SeeTable I. TABLE I Proteins that did Proteins that expressed well notexpress well glucoamylase Human DPPIV alpha-amylase NEP stachybotryslaccase A stachybotrys laccase B human trypsin SCCE bovine prochymosinHer2 antibodies light chain

Selected codons that were not used or not used often in the expressedproteins will be changed to codons that were used often. Therefore, weonly changed a subset of codons.

B. Nucleic Acid Constructs/Expression Vectors.

Natural or synthetic polynucleotide fragments encoding a proteaseinhibitor (“PI-encoding nucleic acid sequences”) may be incorporatedinto heterologous nucleic acid constructs or vectors, capable ofintroduction into, and replication in, a filamentous fungal cell. Thevectors and methods disclosed herein are suitable for use in host cellsfor the expression of a protease inhibitor and variants thereof. Anyvector may be used as long as it is replicable and viable in the cellsinto which it is introduced. Large numbers of suitable vectors andpromoters are known to those of skill in the art, and are commerciallyavailable. Appropriate cloning and expression vectors for use infilamentous fungal cells are also described in Sambrook et al., 1989,and Ausubel F M et al., 1989, expressly incorporated by referenceherein. The appropriate DNA sequence may be inserted into a plasmid orvector (collectively referred to herein as “vectors”) by a variety ofprocedures. In general, the DNA sequence is inserted into an appropriaterestriction endonuclease site(s) by standard procedures. Such proceduresand related sub-cloning procedures are deemed to be within the scope ofknowledge of those skilled in the art.

Appropriate vectors are typically equipped with a selectablemarker-encoding nucleic acid sequence, insertion sites, and suitablecontrol elements, such as termination sequences. The vector may compriseregulatory sequences, including, for example, non-coding sequences, suchas introns and control elements, i.e., promoter and terminator elementsor 5′ and/or 3′ untranslated regions, effective for expression of thecoding sequence in host cells (and/or in a vector or host cellenvironment in which a modified soluble protein coding sequence is notnormally expressed), operably linked to the coding sequence. Largenumbers of suitable vectors and promoters are known to those of skill inthe art, many of which are commercially available and/or are describedin Sambrook, et al., (supra).

Exemplary promoters include both constitutive promoters and induciblepromoters, examples of which include a CMV promoter, an SV40 earlypromoter, an RSV promoter, an EF-1α promoter, a promoter containing thetet responsive element (TRE) in the tet-on or tet-off system asdescribed (ClonTech and BASF), the beta actin promoter and themetallothionein promoter that can upregulated by addition of certainmetal salts. In one embodiment of this invention, glaA promoter is used.This promoter is induced in the presence of maltose. Such promoters arewell known to those of skill in the art.

Those skilled in the art are aware that a natural promoter can bemodified by replacement, substitution, addition or elimination of one ormore nucleotides without changing its function. The practice of theinvention encompasses and is not constrained by such alterations to thepromoter.

The choice of promoter used in the genetic construct is within theknowledge of one skilled in the art.

The choice of the proper selectable marker will depend on the host cell,and appropriate markers for different hosts are well known in the art.Typical selectable marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, for example, ampicillin,methotrexate, tetracycline, neomycin (Southern and Berg, J., 1982),mycophenolic acid (Mulligan and Berg, 1980), puromycin, zeomycin, orhygromycin (Sugden et al., 1985) or (b) compliment an auxotrophicmutation or a naturally occurring nutritional deficiency in the hoststrain. In a preferred embodiment, a fungal pyrg gene is used as aselectable marker (Ballance, D. J. et al., 1983, Biochem. Biophys. Res.Commun. 112: 284-289). In another preferred embodiment, a fungal amdSgene is used as a selectable marker (Tilburn, J. et al., 1983, Gene 26:205-221).

A selected PI coding sequence may be inserted into a suitable vectoraccording to well-known recombinant techniques and used to transform acell line capable of PI expression. Due to the inherent degeneracy ofthe genetic code, other nucleic acid sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be used to clone and express a specific protease inhibitor, asfurther detailed above. Therefore it is appreciated that suchsubstitutions in the coding region fall within the sequence variantscovered by the present invention. Any and all of these sequence variantscan be utilized in the same way as described herein for a parentPI-encoding nucleic acid sequence. One skilled in the art will recognizethat differing Pls will be encoded by differing nucleic acid sequences.

Once the desired form of a protease inhibitor nucleic acid sequence,homologue, variant or fragment thereof, is obtained, it may be modifiedin a variety of ways. Where the sequence involves non-coding flankingregions, the flanking regions may be subjected to resection,mutagenesis, etc. Thus, transitions, transversions, deletions, andinsertions may be performed on the naturally occurring sequence.

Heterologous nucleic acid constructs may include the coding sequence foran protease inhibitor, or a variant, fragment or splice variant thereof:(i) in isolation; (ii) in combination with additional coding sequences;such as fusion protein or signal peptide coding sequences, where the PIcoding sequence is the dominant coding sequence; (iii) in combinationwith non-coding sequences, such as introns and control elements, such aspromoter and terminator elements or 5′ and/or 3′ untranslated regions,effective for expression of the coding sequence in a suitable host;and/or (iv) in a vector or host environment in which the PI codingsequence is a heterologous gene.

A heterologous nucleic acid containing the appropriate nucleic acidcoding sequence, as described above, together with appropriate promoterand control sequences, may be employed to transform filamentous fungalcells to permit the cells to express a protease inhibitor or variantthereof.

In one aspect of the present invention, a heterologous nucleic acidconstruct is employed to transfer a PI-encoding nucleic acid sequenceinto a cell in vitro, with established cell lines preferred. Preferably,cell lines that are to be used as production hosts have the nucleic acidsequences of this invention stably integrated. It follows that anymethod effective to generate stable transformants may be used inpracticing the invention.

In one aspect of the present invention, the first and second expressioncassettes may be present on a single vector or on separate vectors.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,and recombinant DNA, which are within the skill of the art. Suchtechniques are explained fully in the literature. See, for example,“Molecular Cloning: A Laboratory Manual”, Second Edition (Sambrook,Fritsch & Maniatis, 1989), “Animal Cell Culture” (R. I. Freshney, ed.,1987); and “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987). All patents, patent applications, articles andpublications mentioned herein, both supra and infra, are herebyexpressly incorporated herein by reference.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes, also within the knowledge of one skilled in theart.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includebacteriophages λ and M13, as well as plasmids such as pBR322 basedplasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST,and LacZ. Epitope tags can also be added to recombinant proteins toprovide convenient methods of isolation, e.g., c-myc.

The elements that are typically included in expression vectors alsoinclude a replicon, a gene encoding antibiotic resistance to permitselection of bacteria that harbor recombinant plasmids, and uniquerestriction sites in nonessential regions of the plasmid to allowinsertion of heterologous sequences. The particular antibioticresistance gene chosen is not critical, any of the many resistance genesknown in the art are suitable.

C. Host Cells and Culture Conditions.

The present invention provides cell lines comprising cells which havebeen modified, selected and cultured in a manner effective to result inexpression of a protease inhibitor and variants thereof.

Examples of parental cell lines which may be treated and/or modified forPI expression include, but are not limited to, filamentous fungal cells.Examples of appropriate primary cell types for use in practicing theinvention include, but are not limited to, Aspergillus and Trichoderma.

Protease inhibitor expressing cells are cultured under conditionstypically employed to culture the parental cell line. Generally, cellsare cultured in a standard medium containing physiological salts andnutrients, such as standard RPMI, MEM, IMEM or DMEM, typicallysupplemented with 5-10% serum, such as fetal bovine serum. Cultureconditions are also standard, e.g., cultures are incubated at 37° C. instationary or roller cultures until desired levels of protease inhibitorexpression are achieved.

Preferred culture conditions for a given cell line may be found in thescientific literature and/or from the source of the cell line such asthe American Type Culture Collection (ATCC; “http://www.atcc.org/”).Typically, after cell growth has been established, the cells are exposedto conditions effective to cause or inhibit the expression of a proteaseinhibitor and variants thereof.

In the preferred embodiments, where a PI coding sequence is under thecontrol of an inducible promoter, the inducing agent, e.g., acarbohydrate, metal salt or antibiotics, is added to the medium at aconcentration effective to induce protease inhibitor expression.

D. Introduction of a Protease Inhibitor-Encoding Nucleic Acid Sequenceinto Host Cells.

The methods of transformation used may result in the stable integrationof all or part of the transformation vector into the genome of thefilamentous fungus. However, transformation resulting in the maintenanceof a self-replicating extra-chromosomal transformation vector is alsocontemplated.

The invention further provides cells and cell compositions which havebeen genetically modified to comprise an exogenously providedPI-encoding nucleic acid sequence. A parental cell or cell line may begenetically modified (i.e., transduced, transformed or transfected) witha cloning vector or an expression vector. The vector may be, forexample, in the form of a plasmid, a viral particle, a phage, etc, asfurther described above. In a preferred embodiment, a plasmid is used totransfect a filamentous fungal cell. The transformations may besequential or by co-transformation.

Various methods may be employed for delivering an expression vector intocells in vitro. Methods of introducing nucleic acids into cells forexpression of heterologous nucleic acid sequences are also known to theordinarily skilled artisan, including, but not limited toelectroporation; nuclear microinjection or direct microinjection intosingle cells; protoplast fusion with intact cells; use of polycations,e.g., polybrene or polyornithine; or PEG. membrane fusion withliposomes, lipofectamine or lipofection-mediated transfection; highvelocity bombardment with DNA-coated microprojectiles; incubation withcalcium phosphate-DNA precipitate; DEAE-Dextran mediated transfection;infection with modified viral nucleic acids; Agrobacterium-mediatedtransfer of DNA; and the like. In addition, heterologous nucleic acidconstructs comprising a PI-encoding nucleic acid sequence can betranscribed in vitro, and the resulting RNA introduced into the hostcell by well-known methods, e.g., by injection.

Following introduction of a heterologous nucleic acid constructcomprising the coding sequence for a protease inhibitor, the geneticallymodified cells can be cultured in conventional nutrient media modifiedas appropriate for activating promoters, selecting transformants oramplifying expression of a PI-encoding nucleic acid sequence. Theculture conditions, such as temperature, pH and the like, are thosepreviously used for the host cell selected for expression, and will beapparent to those skilled in the art.

The progeny of cells into which such heterologous nucleic acidconstructs have been introduced are generally considered to comprise thePI-encoding nucleic acid sequence found in the heterologous nucleic acidconstruct.

E. Fungal Expression

Appropriate host cells include filamentous fungal cells. The“filamentous fungi” of the present invention, which serve both as theexpression hosts and the source of the first and second nucleic acids,are eukaryotic microorganisms and include all filamentous forms of thesubdivision Eumycotina, Alexopoulos, C. J. (1962), IntroductoryMycology, New York: Wiley. These fungi are characterized by a vegetativemycelium with a cell wall composed of chitin, glucans, and other complexpolysaccharides. The filamentous fungi of the present invention aremorphologically, physiologically, and genetically distinct from yeasts.Vegetative growth by filamentous fungi is by hyphal elongation. Incontrast, vegetative growth by yeasts such as S. cerevisiae is bybudding of a unicellular thallus. Illustrations of differences betweenS. cerevisiae and filamentous fungi include the inability of S.cerevisiae to process Aspergillus and Trichoderma introns and theinability to recognize many transcriptional regulators of filamentousfungi (Innis, M. A. et al. (1985) Science, 228, 21-26).

Various species of filamentous fungi may be used as expression hostsincluding the following genera: Aspergillus, Trichoderma, Neurospora,Penicillium, Cephalosporium, Achlya, Phanerochaete, Podospora, Endothia,Mucor, Fusarium, Humicola, and Chrysosporium. Specific expression hostsinclude A. nidulans, (Yelton, M., et al. (1984) Proc. Natl. Acad. Sci.USA, 81, 1470-1474; Mullaney, E. J. et al. (1985) Mol. Gen. Genet. 199,37-45; John, M. A. and J. F. Peberdy (1984) Enzyme Microb. Technol. 6,386-389; Tilburn, et al. (1982) Gene 26, 205-221; Ballance, D. J. etal., (1983) Biochem. Biophys. Res. Comm. 112, 284-289; Johnston, I. L.et al. (1985) EMBO J. 4, 1307-1311)A. niger, (Kelly, J. M. and M. Hynes(1985) EMBO 4, 475-479) A. niger var. awamori, e.g., NRRL 3112, ATCC22342, ATCC 44733, ATCC 14331 and strain UVK 143f, A. oryzae, e.g., ATCC11490, N. crassa (Case, M. E. et al. (1979) Proc. Natl. Acad. Sci. USA76, 5259-5263; Lambowitz U.S. Pat. No. 4,486,553; Kinsey, J. A. and J.A. Rambosek (1984) Molecular and Cellular Biology 4,117-122; Bull, J. H.and J. C. Wooton (1984) Nature 310, 701-704), Trichoderma reesei, e.g.NRRL 15709, ATCC 13631, 56764, 56765, 56466, 56767, and Trichodermaviride, e.g., ATCC 32098 and 32086. A preferred expression host is A.niger var. awamori in which the gene encoding the major secretedaspartyl protease has been deleted. The production of this preferredexpression host is described in U.S. patent application Ser. No. 214,237filed Jul. 1, 1988, expressly incorporated herein by reference.

During the secretion process in fungi, which are eukaryotes, thesecreted protein crosses the membrane from the cytoplasm into the lumenof the endoplasmic reticulum (ER). It is here that the protein folds anddisulphide bonds are formed. Chaperone proteins such as BiP and proteinslike protein disulphide isomerase assist in this process. It is also atthis stage where sugar chains are attached to the protein to produce aglycosylated protein. Sugars are typically added to asparagine residuesas N-linked glycosylation or to serine or threonine residues as O-linkedglycosylation. Correctly folded and glycosylated proteins pass from theER to the Golgi apparatus where the sugar chains are modified and wherethe KEX2 or KEXB protease of yeast and fungi resides. The N-linkedglycosylation added to secreted proteins produced in fungi differs fromthat added by mammalian cells.

Protease inhibitor and variants thereof produced by the filamentousfungal host cells may be either glycosylated or non-glycosylated (i.e.,aglycosylated or deglycosylated). Because the fungal glycosylationpattern differs from that produced by mammalian cells, the proteaseinhibitor may be treated with an enzyme to deglycosylate the proteaseinhibitor Enzymes useful for such N-linked deglycosylation areendoglycosidase H, endoglycosidase F1, endoglycosidase F2,endoglycosidase A, PNGase F, PNGase A, and PNGase At. Enzymes useful forsuch O-linked deglycosylation are exoglycosidases, specificallyalpha-mannosidases (e.g. alpha-Mannosidase (Aspergillus saito,iGKX-5009), alpha(1-2, 3, 6)-Mannosidase (Jack bean, GKX-5010)alpha-Mannosidase/MANase VI (recombinant from Xanthomonas manihoti,GKX80070) all from Glyko (Prozyme), San Leandro, Calif.).

We have surprisingly found that high levels of a protease inhibitor andvariants thereof can be made in fungi when fused to a native secretedprotein. From the information provided above it is clear that theprotease inhibitor and variants thereof would be expected to assemble inthe ER when glucoamylase was still attached to the N-termini. This wouldproduce a large protein of greater than 56 kD. The glucoamylase wouldnot be expected to be cleaved from the desired protein when it passedthrough the Golgi apparatus without further modification.

Using the present inventive methods and host cells, we have attainedsurprising levels of expression. The system utilized herein has achievedlevels of expression and secretion of greater than 0.5 g/l of proteaseinhibitor.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofgene encoding the desired protein. Large batches of transformed cellscan be cultured as described above. Finally, product is recovered fromthe culture using techniques known in the art.

Chaperones

As noted above, the folding and glycosylation of the secretory proteinsin the ER is assisted by numerous ER-resident proteins calledchaperones. The chaperones like Bip (GRP78), GRP94 or yeast Lhs1p helpthe secretory protein to fold by binding to exposed hydrophobic regionsin the unfolded states and preventing unfavourable interactions(Blond-Elguindi et al., 1993, Cell 75: 717-728). The chaperones are alsoimportant for the translocation of the proteins through the ER membrane.The foldase proteins like protein disulphide isomerase (pdi) and itshomologs and prolyl-peptidyl cis-trans isomerase assist in formation ofdisulphide bridges and formation of the right conformation of thepeptide chain adjacent to proline residues, respectively.

In one aspect of the invention the host cells are transformed with anexpression vector encoding a chaperone. The chaperone is selected fromthe group consisting of pdiA and prpA.

Fermentation Parameters

The invention relies on fermentation procedures for culturing fungi.Fermentation procedures for production of heterologous proteins areknown per se in the art. For example, proteins can be produced either bysolid or submerged culture, including batch, fed-batch andcontinuous-flow processes.

Culturing is accomplished in a growth medium comprising an aqueousmineral salts medium, organic growth factors, the carbon and energysource material, molecular oxygen, and, of course, a starting inoculumof one or more particular microorganism species to be employed.

In addition to the carbon and energy source, oxygen, assimilablenitrogen, and an inoculum of the microorganism, it is necessary tosupply suitable amounts in proper proportions of mineral nutrients toassure proper microorganism growth, maximize the assimilation of thecarbon and energy source by the cells in the microbial conversionprocess, and achieve maximum cellular yields with maximum cell densityin the fermentation media.

The composition of the aqueous mineral medium can vary over a widerange, depending in part on the microorganism and substrate employed, asis known in the art. The mineral media should include, in addition tonitrogen, suitable amounts of phosphorus, magnesium, calcium, potassium,sulfur, and sodium, in suitable soluble assimilable ionic and combinedforms, and also present preferably should be certain trace elements suchas copper, manganese, molybdenum, zinc, iron, boron, and iodine, andothers, again in suitable soluble assimilable form, all as known in theart.

The fermentation reaction is an aerobic process in which the molecularoxygen needed is supplied by a molecular oxygen-containing gas such asair, oxygen-enriched air, or even substantially pure molecular oxygen,provided to maintain the contents of the fermentation vessel with asuitable oxygen partial pressure effective in assisting themicroorganism species to grow in a thriving fashion. In effect, by usingan oxygenated hydrocarbon substrate, the oxygen requirement for growthof the microorganism is reduced. Nevertheless, molecular oxygen must besupplied for growth, since the assimilation of the substrate andcorresponding growth of the microorganisms, is, in part, a combustionprocess.

Although the aeration rate can vary over a considerable range, aerationgenerally is conducted at a rate which is in the range of about 0.5 to10, preferably about 0.5 to 7, volumes (at the pressure employed and at25° C.) of oxygen-containing gas per liquid volume in the fermentor perminute. This amount is based on air of normal oxygen content beingsupplied to the reactor, and in terms of pure oxygen the respectiveranges would be about 0.1 to 1.7, or preferably about 0.1 to 1.3,volumes (at the pressure employed and at 25° C.) of oxygen per liquidvolume in the fermentor per minute.

The pressure employed for the microbial conversion process can rangewidely. Pressures generally are within the range of about 0 to 50 psig,presently preferably about 0 to 30 psig, more preferably at leastslightly over atmospheric pressure, as a balance of equipment andoperating cost versus oxygen solubility achieved. Greater thanatmospheric pressures are advantageous in that such pressures do tend toincrease a dissolved oxygen concentration in the aqueous ferment, whichin turn can help increase cellular growth rates. At the same time thisis balanced by the fact that high atmospheric pressures do increaseequipment and operating costs.

The fermentation temperature can vary somewhat, but for filamentousfungi such as Aspergillus niger var. awamori the temperature generallywill be within the range of about 20° C. to 40° C., generally preferablyin the range of about 28° C. to 37° C., depending on the strain ofmicroorganism chosen.

The microorganisms also require a source of assimilable nitrogen. Thesource of assimilable nitrogen can be any nitrogen-containing compoundor compounds capable of releasing nitrogen in a form suitable formetabolic utilization by the microorganism. While a variety of organicnitrogen source compounds, such as protein hydrolysates, can beemployed, usually cheap nitrogen-containing compounds such as ammonia,ammonium hydroxide, urea, and various ammonium salts such as ammoniumphosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride,or various other ammonium compounds can be utilized. Ammonia gas itselfis convenient for large scale operations, and can be employed bybubbling through the aqueous ferment (fermentation medium) in suitableamounts. At the same time, such ammonia can also be employed to assistin pH control.

The pH range in the aqueous microbial ferment (fermentation admixture)should be in the exemplary range of about 2.0 to 8.0. With filamentousfungi, the pH normally is within the range of about 2.5 to 8.0; withAspergillus niger var. awamori, the pH normally is within the range ofabout 4.5 to 5.5. pH range preferences for certain microorganisms aredependent on the media employed to some extent, as well as theparticular microorganism, and thus change somewhat with change in mediaas can be readily determined by those skilled in the art.

While the average retention time of the fermentation admixture in thefermentor can vary considerably, depending in part on the fermentationtemperature and culture employed, generally it will be within the rangeof about 24 to 500 hours, preferably presently about 24 to 400 hours.

Preferably, the fermentation is conducted in such a manner that thecarbon-containing substrate can be controlled as a limiting factor,thereby providing good conversion of the carbon-containing substrate tocells and avoiding contamination of the cells with a substantial amountof unconverted substrate. The latter is not a problem with water-solublesubstrates, since any remaining traces are readily washed off. It may bea problem, however, in the case of non-water-soluble substrates, andrequire added product-treatment steps such as suitable washing steps.

As described above, the time to reach this limiting substrate level isnot critical and may vary with the particular microorganism andfermentation process being conducted. However, it is well known in theart how to determine the carbon source concentration in the fermentationmedium and whether or not the desired level of carbon source has beenachieved.

Although the fermentation can be conducted as a batch or continuousoperation, fed batch operation is generally preferred for ease ofcontrol, production of uniform quantities of products, and mosteconomical uses of all equipment.

If desired, part or all of the carbon and energy source material and/orpart of the assimilable nitrogen source such as ammonia can be added tothe aqueous mineral medium prior to feeding the aqueous mineral mediumto the fermentor.

Each of the streams introduced into the reactor preferably is controlledat a predetermined rate, or in response to a need determinable bymonitoring such as concentration of the carbon and energy substrate, pH,dissolved oxygen, oxygen or carbon dioxide in the off-gases from thefermentor, cell density measurable by light transmittancy, or the like.The feed rates of the various materials can be varied so as to obtain asrapid a cell growth rate as possible, consistent with efficientutilization of the carbon and energy source, to obtain as high a yieldof microorganism cells relative to substrate charge as possible, butmore importantly to obtain the highest production of the desired proteinper unit volume.

In either a batch, or the preferred fed batch operation, all equipment,reactor, or fermentation means, vessel or container, piping, attendantcirculating or cooling devices, and the like, are initially sterilized,usually by employing steam such as at about 121° C. for at least about15 minutes. The sterilized reactor then is inoculated with a culture ofthe selected microorganism in the presence of all the requirednutrients, including oxygen, and the carbon-containing substrate. Thetype of fermentor employed is not critical, though presently preferredis operation under 15L Biolafitte (Saint-Germain-en-Laye, France).

Protein Separations

Once the desired protein is expressed and, optionally, secreted recoveryof the desired protein may be necessary. The present invention providesmethods of separating a desired protein from its fusion analog. It isspecifically contemplated that the methods described herein are usefulfor the separation of proteinase inhibitor and variants from the fusionanalog.

The collection and purification of the desired protein from thefermentation broth can also be done by procedures known per se in theart. The fermentation broth will generally contain cellular debris,including cells, various suspended solids and other biomasscontaminants, as well as the desired protein product, which arepreferably removed from the fermentation broth by means known in theart.

Suitable processes for such removal include conventional solid-liquidseparation techniques such as, e.g., centrifugation, filtration,dialysis, microfiltration, rotary vacuum filtration, or other knownprocesses, to produce a cell-free filtrate. It may be preferable tofurther concentrate the fermentation broth or the cell-free filtrateprior to crystallization using techniques such as ultrafiltration,evaporation or precipitation.

Precipitating the proteinaceous components of the supernatant orfiltrate may be accomplished by means of a salt, e.g., ammonium sulfateor adjust pH to 2 to 3 and then heat treatment of the broth at 80° C.for 2 hours, followed by purification by a variety of chromatographicprocedures, e.g., ion exchange chromatography, affinity chromatographyor similar art recognized procedures.

When the expressed desired polypeptide is secreted the polypeptide maybe purified from the growth media. Preferably the expression host cellsare removed from the media before purification of the polypeptide (e.g.by centrifugation).

When the expressed recombinant desired polypeptide is not secreted fromthe host cell, the host cell is preferably disrupted and the polypeptidereleased into an aqueous “extract” which is the first stage ofpurification. Preferably the expression host cells are collected fromthe media before the cell disruption (e.g. by centrifugation).

The cell disruption may be performed by conventional techniques such asby lysozyme or beta-glucanase digestion or by forcing the cells throughhigh pressure. See (Robert K. Scobes, Protein Purification, Secondedition, Springer-Verlag) for further description of such celldisruption techniques.

The addition of six histidine residues, i.e., a His Tag, to theC-terminus may also aid in the purification of the desired protein andits fusion analog. Use of the His tag as a purification aid is wellknown in the art. See, for example, Hengen (1995) TIBS 20 (7): 285-286.The 6×his-tagged proteins are easily purified using Immobilized Metalion Affinity Chromatography (IMAC).

It is specifically contemplated that protease inhibitors and variantsthereof may be purified from an aqueous protein solution, e.g., wholecell fermentation broth or clarified broth, using a combination ofhydrophobic charge induction chromatography (HCIC). HCIC provided anability to separate the desired protein from the broth and from itsfusion analog.

Utility

For some applications of desired proteins it is of high importance thatthe protease inhibitors are extremely pure, e.g. having a purity of morethan 99%. This is particularly true whenever the desired protein is tobe used as a therapeutic, but is also necessary for other applications.The methods described herein provide a way of producing substantiallypure desired proteins. The desired proteins described herein are usefulin pharmaceutical and personal care compositions.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); kg (kilograms); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); h (hours); min (minutes); sec (seconds); msec(milliseconds); Ci (Curies) mCi (milliCuries); μCi (microCuries); TLC(thin layer achromatography); Ts (tosyl); Bn (benzyl); Ph (phenyl); Ms(mesyl); Et (ethyl), Me (methyl). PI (proteinase inhibitor), BBI(Bowman-Birk inhibitor), STI (Soybean Trypsin inhibitor).

EXAMPLES

The present invention is described in further detail in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed. The attached Figures are meant to be considered asintegral parts of the specification and description of the invention.All references cited are herein specifically incorporated by referencefor all that is described therein. The following examples are offered toillustrate, but not to limit the claimed invention.

Example 1 Cloning of DNA encoding the Soybean Trypsin Inhibitor

This example illustrates the development of an expression vector forSTI.

In general, the gene encoding the desired protein was fused to the DNAencoding the linker region of glucoamylase with an engineered kexBcleavage site (NVISKR) via an NheI restriction enzyme site at theN-terminal and a BstEII restriction enzyme site at the C-terminalfollowing the STI stop codon, TAG. The gene encoding the soybean STI wassynthesized by MCLAB (South San Francisco, Calif.) in vitro as a DNAfragment containing two restriction sites, a kexB cleavage site andthree glycine residues at N-terminal end and six histidine residues atC-terminal end. (SEQ ID NO:3, gene shown in FIG. 2). All PCR-generatedDNA fragments used herein were initially cloned into the pCRII-TOPOvector (Invitrogen, Carlsbad, Calif.). E. coli [One Shot® TOP10 cellsfrom Invitrogen], was used for routine plasmid isolation and plasmidmaintenance. The NheI and BstEII sites were used to excise the PCRproduct from the pCRII-TOPO vector, and the resulting DNA fragment wasthen ligated into the expression vector, pSL1180-GAMpR-2 (see FIG. 5)The expression vector, pSL1180-GAMpR2, contains the Aspergillus nigerglucoamylase promoter, the glucoamylase catalytic domain and theterminator region. The expression plasmid also contains the A. nigerpyrG gene as the selection marker. Thus, detection of transformants withthe expression cassette is by growth on uridine-deficient medium.

The gene encoding the STI peptide (for amino acid sequence: FIG. 4A, SEQID NO:10; for nucleotide sequence: FIG. 2 and SEQ ID NO:6) wassynthesized and cloned into pCRII-TOPO vector (Invitrogen) by MCLAB. TheNheI to BstEII fragment was release from the plasmid by restrictiondigestion and the DNA fragment was extracted from an agarose gel andcloned into pSLGAMpR2, a glucoamylase-chymosin expression vector whichis described in detail in WO 9831821. to create expression plasmidpSLGAMpR2-SBTI/nonopti (Q110).

The expression plasmid was transformed into dgr246ΔGAP:pyr2-. Thisstrain is derived from strain dgr246 P2 which has the pepA gene deleted,is pyrG minus and has undergone several rounds of mutagenesis andscreening or selection for improved production of a heterologous geneproduct (Ward, M. et al., 1993, Appl. Microbiol. Biotech. 39: 738-743and references therein). To create strain dgr246ΔGAP:pyr2- the glaA(glucoamylase) gene was deleted in strain dgr246 P2 using exactly thesame deletion plasmid (pΔGAM NB-Pyr) and procedure as reported byFowler, T. et al (1990) Curr. Genet. 18: 537-545. Briefly, the deletionwas achieved by transformation with a linear DNA fragment having glaAflanking sequences at either end and with part of the promoter andcoding region of the glaA gene replaced by the Aspergillus nidulans pyrGgene as selectable marker. Transformants in which the linear fragmentcontaining the glaA flanking sequences and the pyrG gene had integratedat the chromosomal glaA locus were identified by Southern blot analysis.This change had occurred in transformed strain dgr246ΔGAP. Spores fromthis transformant were plated onto medium containing fluoroorotic acidand spontaneous resistant mutants were obtained as described by vanHartingsveldt, W. et al. (1987) Mol. Gen. Genet. 206: 71-75. One ofthese, dgr246ΔGAP:pyr2-, was shown to be a uridine auxotroph strainwhich could be complemented by transformation with plasmids bearing awild-type pyrG gene.

The Aspergillus transformation protocol was a modification of theCampbell method (Campbell et at. (1989). Curr. Genet. 16: 53-56). Allsolutions and media were either autoclaved or filter sterilized througha 0.2 micron filter. Spores of A. niger var. awamori were harvested fromcomplex media agar (CMA) plates. CMA contained 20 g/l dextrose, 20 g/lDifco Brand malt extract, 1 g/l Bacto Peptone, 20 g/l Bacto agar, 20ml/l of 100 mg/ml arginine and 20 ml/l of 100 mg/ml uridine. An agarplug of approximately 1.5 cm square of spores was used to inoculate 100mls of liquid CMA (recipe as for CMA except that the Bacto agar wasomitted). The flask was incubated at 37° C. on a shaker at 250-275 rpm,overnight. The mycelia were harvested through sterile Miracloth(Calbiochem, San Diego, Calif., USA) and washed with 50 mls of SolutionA (0.8M MgSO₄ in 10 mM sodium phosphate, pH 5.8). The washed myceliawere placed in a sterile solution of 300 mg of beta-D-glucanase(Interspex Products, San Mateo, Calif.) in 20 mls of solution A. Thiswas incubated at 28° to 30° C. at 200 rpm for 2 hour in a sterile 250 mlplastic bottle (Corning Inc, Corning, N.Y.). After incubation, thisprotoplasting solution was filtered through sterile Miracloth into asterile 50 ml conical tube (Sarstedt, USA). The resulting liquidcontaining protoplasts was divided equally amongst two 50 ml conicaltubes. Forty ml of solution B (1.2 M sorbitol, 50 mM CaCl₂, 10 mM Tris,pH 7.5) were added to each tube and centrifuged in a table top clinicalcentrifuge (Damon IEC HN SII centrifuge) at full speed for 5 minutes.The supernatant from each tube was discarded and 20 ms of fresh solutionB was added to one tube, mixed, then poured into the next tube until allthe pellets were resuspended. The tube was then centrifuged for 5minutes. The supernatant was discarded, 20 mls of fresh solution B wasadded, the tube was centrifuged for 5 minutes. The wash occurred onelast time before resuspending the washed protoplasts in solution B at adensity of 0.5-1.0×10⁷ protoplasts/100 ul. To each 100 ul of protoplastsin a sterile 15 ml conical tube (Sarstedt, USA), 10 ul of thetransforming plasmid DNA was added. To this, 12.5 ul of solution C (50%PEG 4000, 50 mM CaCl₂, 10 mM Tris, pH 7.5) was added and the tube wasplaced on ice for 20 minutes. One ml of solution C was added and thetube was removed from the ice to room temperature and shaken gently. Twoml of solution B was added immediately to dilute solution C. Thetransforming mix was added equally to 3 tubes of melted MMS overlay (6g/l NaNO₃, 0.52 g/l KCl, 1.52 g/l KH₂PO₄, 218.5 g/l D-sorbitol, 1.0 ml/ltrace elements-LW, 10 g/l SeaPlaque agarose (FMC Bioproducts, Rook1and,Me., USA) 20 ml/l 50% glucose, 2.5 ml/l 20% MgSO₄.7H₂O, pH to 6.5 withNaOH) that were stored in a 45° C. water bath. Trace elements-LWconsisted of 1 g/l FeSO₄.7H₂O, 8.8 g/l ZnSO₄.7H₂O, 0.4 g/l CuSO₄.5H₂O,0.15 g/l MnSO₄.4H₂O, 0.1 g Na₂B₄O₇.10H₂O, 50 mg/l (NH₄)₆Mo₇O₂₄.4H₂O, 250mls H₂O, 200 ul/l concentrated HCl. The melted overlays with thetransformation mix were immediately poured onto 3 MMS plates (same asMMS overlay recipe with the exception of 20 g/l of Bacto agar instead of10 g/l of SeaPlaque agarose) that had been supplemented with 333ul/plate of 100 mg/ml of arginine added directly on top of the agarplate. After the agarose solidified, the plates were incubated at 30° C.until transformants grew.

The sporulating transformants were picked off with a sterile toothpickonto a plate of minimal media+glucose (MM). MM consisted of 6 g/l NaNO₃,0.52 g/l KCl, 1.52 g/l KH₂PO₄, 1 ml/l Trace elements-LW, 20 g/l Bactoagar, pH to 6.5 with NaOH, 25 ml/I of 40% glucose, 2.5 ml/l of 20%MgSO₄.7H₂O and 20 ml/l of 100 mg/ml arginine. Once the transformantsgrew on MM they were transferred to CMA plates.

A 1.5 cm square agar plug from a plate culture of each transformant wasadded to 50 mls, in a 250 ml shake flask, of production medium calledPromosoy special. This medium had the following components: 70 g/lsodium citrate, 15 g/l (NH₄)₂ SO₄, 1 g/l NaH₂PO₄.H₂O, 1 g/l MgSO₄, 1 mlTween 80, pH to 6.2 with NaOH, 2 ml/l Mazu DF60-P, 45 g/l Promosoy 100(Central Soya, Fort Wayne, Ind.), 120 g/l maltose. The production mediaflasks were incubated at 30° C., 200 rpm for 5 days and supernatantsamples were harvested. Transformants were assayed for proteinproduction on SDS gel to select the transformants based on the amount ofprotein produced. Broth from the top transformants were assayed forTrypsin or chymotrypsin inhibition activity.

A 1.5 cm square agar plug from a plate culture of each transformant wasalso added to 50 mis, in a 250 ml shake flask, of production mediumcalled modified CSS. This medium had the following components: 50 g/lCorn Streep Solids, 1 g/l NaH2PO4*H2O, 0.5 g/l MgSO4 (anhydrous), 50 g/lStaley 7350 (55%) and 8 g/l Na Citrate. The production media flasks wereincubated at 36° C., 200 rpm for 3 days and supernatant samples wereharvested and assayed for protein production on SDS gel. Broth from thetop transformants were assayed for Trypsin or Chymotrypsin inhibitionactivity.

Example 2 Codon Optimization of the DNA Encoding the Soybean TrypsinInhibitor

The following example details how the STI-encoding DNA was altered foroptimized expression in a filamentous fungi.

The codons from the synthetic gene (the starting material in Example 1that was synthesized by MCLAB) were then optimized according to thecodon usage of highly expressed proteins in Aspergillus. Basically,proteins that expressed well such as glucoamylase, alpha-amylase andprochymosin were compared to proteins that did not express well inAspergillus such as human NEP and DPP4. See Table I. The codon usagetable for both types of protein expressions in Aspergillus is in TableII. TABLE II glucoamylase NEP bovine Her2 Stachybotrys stachybotrysw/out starch Codon AA coding DPP4 SCCE prochymosin chymotrypsin Lightchain oxidase B oxidase A binding domain gca* Ala(A) 19 15 1 0 1 1 2 410 gcc Ala(A) 12 6 8 15 17 8 19 23 18 gcg Ala(A) 1 2 0 1 1 2 1 0 9 gcuAla(A) 18 12 2 1 3 3 25 21 20 --- Ala(A) 50 35 11 17 22 14 47 48 57 aga*Arg(R) 15 16 3 1 0 2 2 2 1 agg Arg(R) 5 8 4 6 4 1 4 5 1 cga Arg(R) 6 1 01 0 0 7 3 3 cgc Arg(R) 2 1 4 1 1 2 12 11 6 cgg Arg(R) 1 3 0 0 0 0 0 0 2cgu Arg(R) 4 1 0 0 1 4 7 8 4 --- Arg(R) 33 30 11 9 6 9 32 29 17 aacAsn(N) 20 16 1 11 6 5 26 27 17 aau* Asn(N) 36 23 9 4 3 1 5 6 6 ---Asn(N) 56 39 10 15 9 6 31 33 23 gac Asp(D) 13 16 10 20 14 5 24 20 17gau* Asp(D) 28 27 4 2 1 5 18 19 18 --- Asp(D) 41 43 14 22 15 10 42 39 35ugc Cys(C) 7 4 10 3 8 4 1 0 6 ugu Cys(C) 5 8 2 3 2 1 0 1 2 --- Cys(C) 1212 12 6 10 5 1 1 8 caa* Gln(Q) 14 15 3 1 1 3 2 6 4 cag Gln(Q) 17 15 7 248 12 14 13 11 --- Gln(Q) 31 30 10 25 9 15 16 19 15 gaa* Glu(E) 36 31 4 21 1 2 4 7 gag Glu(E) 17 9 2 12 5 8 38 42 11 --- Glu(E) 53 40 6 14 6 9 4046 18 gga* Gly(G) 15 20 5 1 1 2 10 10 5 ggc Gly(G) 12 7 11 15 14 5 17 2020 ggg Gly(G) 7 6 1 12 7 0 0 1 4 ggu Gly(G) 7 7 4 3 1 4 17 11 12 ---Gly(G) 41 40 21 31 23 11 44 42 41 cac His(H) 5 8 5 4 3 2 11 13 4 cauHis(H) 4 11 2 2 0 1 2 4 0 --- His(H) 9 19 7 6 3 3 13 17 4 aua Ile(I) 913 1 1 1 0 0 0 0 auc Ile(I) 10 12 3 19 9 5 19 16 8 auu* Ile(I) 26 22 2 22 1 6 12 11 --- Ile(l) 45 47 6 22 12 6 25 28 19 cua* Leu(L) 5 6 0 0 0 00 0 2 cuc Leu(L) 7 8 8 5 4 3 13 18 16 cug Leu(L) 9 15 10 23 15 10 14 1615 cuu Leu(L) 15 7 0 1 0 0 11 12 3 uua* Leu(L) 7 10 1 0 0 0 0 0 0 uug*Leu(L) 16 9 1 0 0 1 5 3 6 --- Leu(L) 59 55 20 29 19 14 43 49 42 aaa*Lys(K) 32 27 3 6 1 5 0 0 0 aag Lys(K) 17 10 13 9 14 9 7 19 11 --- Lys(K)49 37 16 15 15 14 7 19 11 aug Met(M) 14 14 6 8 2 1 17 12 3 --- Met(M) 1414 6 8 2 1 17 12 3 uuc Phe(F) 12 14 3 13 6 9 24 21 17 uuu* Phe(F) 16 171 6 1 0 3 6 2 --- Phe(F) 28 31 4 19 7 9 27 27 19 cca* Pro(P) 9 14 4 1 02 1 6 0 ccc Pro(P) 6 2 7 11 8 6 20 17 8 ccg Pro(P) 0 1 1 3 0 2 4 2 6 ccuPro(P) 7 10 2 1 5 2 21 16 3 --- Pro(P) 22 27 14 16 13 12 46 41 17 agcSer(S) 7 12 2 13 6 14 8 7 19 agu* Ser(S) 7 14 2 4 1 1 2 1 10 uca* Ser(S)11 17 3 1 0 0 2 2 2 ucc Ser(S) 7 10 9 9 14 11 11 8 16 ucg Ser(S) 0 1 1 40 2 4 3 11 ucu Ser(S) 11 10 1 4 2 5 9 8 15 --- Ser(S) 43 64 18 35 23 3336 29 73 uaa Ter(.) 0 0 1 0 0 0 0 1 0 uag Ter(.) 0 1 0 0 0 0 0 0 0 ugaTer(.) 1 0 0 0 1 1 0 0 0 --- Ter(.) 1 1 1 0 1 1 0 1 0 aca* Thr(T) 10 211 5 4 2 2 1 4 acc Thr(T) 9 6 9 13 13 14 12 16 28 acg Thr(T) 1 1 3 2 0 21 1 8 acu Thr(T) 10 17 5 4 0 3 16 12 14 --- Thr(T) 30 45 18 24 17 21 3130 54 ugg Trp(W) 14 20 5 4 8 2 11 14 15 --- Trp(W) 14 20 5 4 8 2 11 1415 uac Tyr(Y) 11 26 4 17 2 9 21 24 16 uau* Tyr(Y) 22 30 0 5 0 2 4 4 6--- Tyr(Y) 33 56 4 22 2 11 25 28 22 gua* Val(V) 5 7 0 2 0 1 0 2 2 gucVal(V) 9 12 6 7 10 10 17 25 13 gug Val(V) 13 14 10 14 14 4 8 6 14 guuVal(V) 10 11 2 3 0 1 21 12 5 --- Val(V) 37 44 18 26 24 16 46 45 34 nnn???(X) 0 0 0 0 0 0 3 0 0 TOTAL 701 729 232 365 246 222 583 597 527

It is evident that many codons were not used or not used as often in thegenes that expressed well. These codons were found much more frequentlyin those genes that were not expressed well (indicated with an asteriskin Table II). In the STI gene, we identified several such codons thatwere not used or not used often by other well expressed proteins and thecodons were changed to the codons that are used more often in wellexpressed proteins. See Tables III and IV. TABLE III Codon usage forwild type STI: (without three glycine residues and six histidineresidues and the stop codon) gca Ala(A) 4 # cag Gln(Q) 2 # uug Leu(L) 3# uaa Ter(.)  0 gcc Ala(A) 2 # --- Gln(Q) 5 # --- Leu(L) 15 # uag Ter(.) 0 gcg Ala(A) 0 # gaa Glu(E) 7 # aaa Lys(K) 7 # uga Ter(.)  0 gcu Ala(A)2 # gag Glu(E) 6 # aag Lys(K) 3 # --- Ter(.)  0 --- Ala(A) 8 # ---Glu(E) 13 # --- Lys(K) 10 # aca Thr(T)  3 aga Arg(R) 4 # gga Gly(G) 6 #aug Met(M) 2 # acc Thr(T)  2 agg Arg(R) 1 # ggc Gly(G) 2 # --- Met(M) 2# acg Thr(T)  1 cga Arg(R) 1 # ggg Gly(G) 3 # uuc Phe(F) 4 # acu Thr(T) 1 cgc Arg(R) 1 # ggu Gly(G) 5 # uuu Phe(F) 5 # --- Thr(T)  7 cgg Arg(R)0 # --- Gly(G) 16 # --- Phe(F) 9 # ugg Trp(W)  2 cgu Arg(R) 2 # cacHis(H) 0 # cca Pro(P) 4 # --- Trp(W)  2 --- Arg(R) 9 # cau His(H) 2 #ccc Pro(P) 0 # uac Tyr(Y)  0 aac Asn(N) 4 # --- His(H) 2 # ccg Pro(P) 1# uau Tyr(Y)  4 aau Asn(N) 5 # aua Ile(I) 3 # ccu Pro(P) 5 # --- Tyr(Y) 4 --- Asn(N) 9 # auc Ile(I) 5 # --- Pro(P) 10 # gua Val(V)  0 gacAsp(D) 3 # auu Ile(I) 6 # agc Ser(S) 1 # guc Val(V)  0 gau Asp(D) 14 #--- Ile(I) 14 # agu Ser(S) 1 # gug Val(V)  8 --- Asp(D) 17 # cua Leu(L)1 # uca Ser(S) 3 # guu Val(V)  6 ugc Cys(C) 1 # cuc Leu(L) 3 # uccSer(S) 1 # --- Val(V)  14 ugu Cys(C) 3 # cug Leu(L) 2 # ucg Ser(S) 1 #nnn ???(X)  0 --- Cys(C) 4 # cuu Leu(L) 5 # ucu Ser(S) 4 # TOTAL 181 caaGln(Q) 3 # uua Leu(L) 1 # --- Ser(S) 11 #

TABLE IV Codon usage for A. niger codon optimized STI I: (without threeglycine residues and six histidine residues and the stop codon): gcaAla(A) 4 # cag Gln(Q) 2 # uug Leu(L) 0 # uaa Ter(.)  0 gcc Ala(A) 2 #--- Gln(Q) 5 # --- Leu(L) 15 # uag Ter(.)  0 gcg Ala(A) 0 # gaa Glu(E) 7# aaa Lys(K) 7 # uga Ter(.)  0 gcu Ala(A) 2 # gag Glu(E) 6 # aag Lys(K)3 # --- Ter(.)  0 --- Ala(A) 8 # --- Glu(E) 13 # --- Lys(K) 10 # acaThr(T)  3 aga Arg(R) 0 # gga Gly(G) 6 # aug Met(M) 2 # acc Thr(T)  2 aggArg(R) 1 # ggc Gly(G) 3 # --- Met(M) 2 # acg Thr(T)  1 cga Arg(R) 1 #ggg Gly(G) 3 # uuc Phe(F) 4 # acu Thr(T)  1 cgc Arg(R) 5 # ggu Gly(G) 4# uuu Phe(F) 5 # --- Thr(T)  7 cgg Arg(R) 0 # --- Gly(G) 16 # --- Phe(F)9 # ugg Trp(W)  2 cgu Arg(R) 2 # cac His(H) 0 # cca Pro(P) 0 # ---Trp(W)  2 --- Arg(R) 9 # cau His(H) 2 # ccc Pro(P) 0 # uac Tyr(Y)  4 aacAsn(N) 4 # --- His(H) 2 # ccg Pro(P) 1 # uau Tyr(Y)  0 aau Asn(N) 5 #aua Ile(I) 0 # ccu Pro(P) 9 # --- Tyr(Y)  4 --- Asn(N) 9 # auc Ile(I) 8# --- Pro(P) 10 # gua Val(V)  0 gac Asp(D) 3 # auu Ile(I) 6 # agc Ser(S)1 # guc Val(V)  0 gau Asp(D) 14 # --- Ile(I) 14 # agu Ser(S) 1 # gugVal(V)  8 --- Asp(D) 17 # cua Leu(L) 1 # uca Ser(S) 3 # guu Val(V)  6ugc Cys(C) 1 # cuc Leu(L) 3 # ucc Ser(S) 1 # --- Val(V)  14 ugu Cys(C) 3# cug Leu(L) 6 # ucg Ser(S) 1 # nnn ???(X)  0 --- Cys(C) 4 # cuu Leu(L)5 # ucu Ser(S) 4 # TOTAL 181 caa Gln(Q) 3 # uua Leu(L) 0 # --- Ser(S) 11#

The optimized DNA was synthesized by MCLAB (South San Francisco) invitro as a DNA fragment containing three restriction sites (NheI at 5′end of gene and XhoI and BstEII at the 3′ end), a kexB cleavage site andthree glycine residues at N-terminal end and six histidine residues atC-terminal (SEQ I.D. NO:3). This optimized gene was cloned into apCRII-TOPO vector. Following the procedures described in Example 1above, the NheI to BstEII fragment was released from the plasmid byrestriction digestion and the DNA fragment was purified on and extractedfrom an agarose gel and cloned into pSLGAMpR2 to create expressionplasmid pSLGAMpR2-SBTI (Q107).

The expression plasmid was transformed into dgr246ΔGAP:pyr2. Thetransformation and shake flask testing of transformants were as inExample 1. Thirty one transformants were assayed and SDS gel was used tocheck the level of protein expression. Broth from the top sixtransformants were assayed for trypsin inhibition activity.

Example 3 Expression of the Bowman-Birk Inhibitor and its Variants inAspergillus

a. BBI Fusion to Glucoamylase with kexB Site and with Three Glycine atN-Terminal End and Six Histidine Residues at C-Terminal:

Following procedures described in Example 2 above, the BBI-encoding DNAwas optimized and used for this Example. The DNA was synthesized byMCLAB in vitro as a DNA fragment containing three restriction sites(NheI at 5′ end of gene and XhoI and BstEII at the 3′ end), a kexBcleavage site and three glycine residues at N-terminal and six histidineresidues at C-terminal. (SEQ ID NO:54). It was cloned into pCRII-TOPOvector Invitrogen. Following procedures described in Example 1 above,the NheI to BstEII fragment was released from the plasmid by restrictiondigestion and the DNA fragment was extracted from agarose gel and clonedinto pSLGAMpR2 to create expression plasmid pSLGAMpR2-BBlkex+(Q104). Theexpression plasmid was transformed into dgr246ΔGAP:pyr2. Thetransformation and shake flask testing of transformants were same asExample 1. Twenty-eight transformants were generated and twenty-fivetransformants were assayed in shake flask. The SDS gel was used to checkthe level of protein expression. Broth from the top transformants wereassayed for trypsin or chymotrypsin inhibition activity.

b. BBI Fusion to Glucoamylase with Six Histidine Residues at C-Terminal:

Following procedures described in Example 2 above, the BBI-encoding DNAwas optimized and used for this Example. The DNA was synthesized byMCLAB in vitro as a DNA fragment containing three restriction sites(NheI at 5′ end of gene and XhoI and BstEII at the 3′ end) and sixhistidine residues at C-terminal. (SEQ ID NO:42:GCTAGCGACGATGAGAGCTCTMGCCCTGTTGCGATCAGTGCGCGTGTACCAAATCGMCCCTCCGCAGTGTCGCTGCTCCGATATGCGTCTGAATTCCTGTCATAGCGCATGCMGAGCTGTATCTGCGCCCTGAGCTACCCCGCGCAGTGTTTCTGCGTCGACATCACGGACTTCTGCTACGAGCCGTGTMGCCCAGCGAGGACGATMGGAGAACCATCATCACCATCACCATTAGCTCGAGGGTGACC). It was cloned into pCRII-TOPO vector.Following procedures described in Example 1 above, the NheI to BstEIIfragment was release from the plasmid by restriction digestion, purifiedand extracted from agarose gel, and cloned into pSLGAMpR2 to createexpression plasmid pSLGAMpR2-BBlkex-(Q105). The expression plasmid wastransformed into dgr246ΔGAP:pyr2. The transformation and shake flasktesting of transformants were same as example 1. Thirty-eighttransformants were generated and twenty-five transformants were assayedin shake flask. The SDS gel was used to check the level of proteinexpression. Broth from the top transformants were assayed for trypsin orchymotrypsin inhibition activity.

c. BBI Fusion to Glucoamylase with kexB Site and Three Glycine Residuesat N-Terminal End:

The plasmid DNA, synthesized by MCLAB in vitro (SEQ ID NO:5) which wascloned into pCRII-TOPO vector, was used as DNA template for PCRamplification. Two primers were designed: 5′ GGG CTA GCA ACG TCA TCT CCAAG 3′ (SEQ ID NO:43) and 5′ GGG GTC ACC TAG TTC TCC TTA TCG TCC TCG CTG3′ (SEQ ID NO:44). The DNA was amplified in the presence of the primersunder the following conditions: The DNA was diluted 10 to 100 fold withTris-EDTA buffer. Ten microliter of diluted DNA was added to thereaction mixture which contained 0.2 mM of each nucleotide (A, G. C andT), 1× reaction buffer, 0.5 to 0.6 microgram of primer 1 (SEQ ID NO:43)and primer 2 (SEQ ID NO:44) in a total of 100 microliter reaction in aneppendorf tube. After heating the mixture at 100° C. for 5 minutes, 2.5units of Taq DNA polymerase were added to the reaction mix. The PCRreaction was performed at 95° C. for 1 minute, the primer was annealedto the template at 50° C. for 1 minute and extension was done at 72° C.for 1 minute. This cycle was repeated 30 times with an additional cycleof extension at 68° C. for 7 minutes before stored at 4° C. for furtheruse. The PCR fragment detected by agarose gel was then cloned into theplasmid vector pCRII-TOPO (Invitrogen). The resulting PCR fragmentcontains identical sequence as SEQ ID NO:54, except the nucleotidesencoding the six histidine residues and the XhoI restriction site wereremoved. Following procedures described in Example 1 above, the PCRfragment was digested with restriction enzymes NheI and BstEII. Thedigested DNA fragment was precipitated by ethanol and cloned intopSLGAMpR2 to create expression plasmid pSLGAMpR2-BBI without histag(Q108). The expression plasmid was transformed into dgr246ΔGAP:pyr2. Thetransformation and shake flask testing of transformants were same asdescribed in Example 1. Fifty-seven transformants were generated andtwenty-five transformants were assayed in shake flask. The SDS gel wasused to check the level of protein expression. Broth from the toptransformants were assayed for trypsin or chymotrypsin inhibitionactivity.

d. BBI Fusion to Glucoamylase with kexB Site:

The plasmid DNA, synthesized by MCLAB in vitro (SEQ ID NO:1) which wascloned into pCRII-TOPO vector, was used as DNA template for PCRamplification. Two primers were designed: 5′ GGG GTC ACC TAG TTC TCC TTATCG TCC TCG CTG 3′ (SEQ ID NO:44) and 5′ GGG CTA GCA ACG TCA TCT CCA AGCGCG ACG ATG AGA GCT CTA AG 3′ (SEQ ID NO:45). The resulting PCR fragmentcontains identical sequence as SEQ ID NO:54 (FIG. 1C), except thenucleotides encoding the three glycine residues and six histidineresidues and XhoI restriction site were removed. Following proceduresdescribed in Example 1 above, the PCR fragment was digested withrestriction enzymes NheI and BstEII. The digested DNA fragment wasprecipitated by ethanol and cloned into pSLGAMpR2 to create expressionplasmid pSLGAMpR2-BBI without 3G and histag (Q109). The expressionplasmid was transformed into dgr246ΔGAP:pyr2. The transformation andshake flask testing of transformants were same as Example 1. One hundredand twenty-seven transformants were generated and forty-twotransformants were assayed in shake flask. The SDS gel was used to checkthe level of protein expression. Broth from the top transformants wereassayed for trypsin or chymostrypsin inhibition activity.

Example 4 Expression of the Bowman-Birk Inhibitor and its Variants (LoopReplacement by Other Binders) in Aspergillus

Variant sequences were introduced into one or both loops of BBI usingstandard procedures known in the art. Variant sequences were determinedby panning a commercially available phage peptide library PhD C7C (NewEngland Biolabs, Beverly, Mass.) against target proteins or substratesfor 3 rounds according to the manufacturers instructions, or usingsequences with known activity. In the sequences provided below, thealterations introduced into the loop nucleotide sequence is indicated bylower case nucleotides.

a. BBI with a-VEGF (CK37281) in Loop I

The plasmid DNA, synthesized by MCLAB in vitro (SEQ ID NO:1) which wascloned into pCRII-TOPO vector, was used as DNA template for PCRamplification. Two primers were designed:

-   5′ GTTGCGATCAGTGCGCGTGTtacaatctgtatggctggaccTGTCGCTGCT 3′ (SEQ ID    NO:46) and-   5′CGCATATCGGAGCAGCGACAggtccagccatacagattgtaACACGCGCAC 3′. (SEQ ID    NO:47)    to introduce a peptide sequence that binds to VEGF (denoted a-VEGF)    to inhibit VEGF function. PCR was performed by heating mixture at    94° C. for 2 min, then 30 cycles of reaction at 94° C. for 30    second, 63° C. for 30 second and 72° C. for 30 second. After 30    cycles, the mixture was incubated at 72° C. for 4 min before it was    stored at 4° C. The replacement binding loop was verified by DNA    sequencing. The NheI to BstEII DNA fragment was released from    plasmid by restriction digestion, purified and cloned into pSLGAMpR2    to create expression plasmid pSLGAMpR2-BBI (CK37281) in loop1    (Q117). The expression plasmid was transformed into dgr246ΔGAP:pyr2,    The transformation and shake flask testing of transformants were    same as in Example 1. More than thirty transformants were generated    and forty-two transformants were assayed in shake flask. The SDS gel    was used to check the level of protein expression.    b. BBI with a-VEGF (CK37281) Peptide in Loop II:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures asdescribed in example above, except the following two primers were used:5′ CATGCAAGAGCTGTATCTGCtacaatctgta (SEQ ID NO: 48)tggctggaccCAGTGTTTCTG3′ 5′ GATGTCGACGCAGAAACACTGggtccagcca (SEQ ID NO:49) tacagattgtaGCAGATACAG3′.c. BBI with a-VEGF (CK37281) Peptide in Loop I and II:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures,except the following four primers were used: 5′GTTGCGATCAGTGCGCGTGTtacaatctgta (SEQ ID NO: 46) tggctggaccTGTCGCTGCT 3′5′ CGCATATCGGAGCAGCGACAggtccagccat (SEQ ID NO: 47) acagattgtaACACGCGCAC3′ 5′ CATGCAAGAGCTGTATCTGCtacaatctgta (SEQ ID NO: 48)tggctggaccCAGTGTTTCTG3′ 5′ GATGTCGACGCAGAAACACTGggtccagcca (SEQ ID NO:49) tacagattgtaGCAGATACAG3′.d. BBI with a-Complement Protein c2 Peptide in Loop 1:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures,except the following two primers were used to introduce a peptidesequence that binds to c2 (denoted a-c2) to inhibit c2 function:5′GCGATCAGTGCAGCTGTagctgcggcaggaag (SEQ ID NO: 50)atccccatccagtgcTGTCGCTGCTCCGATATGC GTC3′5′GAGCAGCGACAgcactggatggggatcttcct (SEQ ID NO: 51)gccgcagctACAGCTGCACTGATCGCAACAGGGC TTA3′e. BBI with a-Complement Protein c3 Peptide in Loop I:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures,except the following two primers were used to introduce a peptidesequence that binds to c3 (denoted a-c3) to inhibit c3 function: 5′GCGATCAGTGCGGCTGTgccaggagcaacct (SEQ ID NO: 52)cgacgagTGTCGCTGCTCCGATATGCGTC 3′ 5′ GAGCAGCGACActcgtcgaggttgctcctgg (SEQID NO: 53) cACAGCCGCACTGATCGCAACAGGGCTTA 3′f. BBI with a-Complement Protein c4 Peptide in Loop I:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures,except the following two primers were used to introduce a peptidesequence that binds to c4 (denoted a-c4) to inhibit c4 function: 5′GCGATCAGTGCGCGTGTcagagggccctccc (SEQ ID NO: 55)catcctcTGTCGCTGCTCCGATATGCGTC 3′ 5′ GAGCAGCGACAgaggatggggagggccctct (SEQID NO: 56) gACACGCGCACTGATCGCAACAGGGCTTA 3′

-   -   g. BBI with a-Complement Protein c5 Peptide in Loop I:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures,except the following two primers were used to introduce a peptidesequence that binds to c5 (denoted a-c5) to inhibit c5 function: 5′GCGATCAGTGCCAGTGTggcaggctccacat (SEQ ID NO: 57)gaagaccTGTCGCTGCTCCGATATGCGTC 3′ 5′ GAGCAGCGACAggtcttcatgtggagcctgc (SEQID NO: 58) cACACTGGCACTGATCGCAACAGGGCTTAGA 3′

-   -   h. BBI with a-Human Complement Factor B Peptide in Loop I:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures,except the following two primers were used to introduce a peptidesequence that binds to Factor B (denoted a-Factor B) to inhibit Factor Bfunction: 5′ GCGATCAGTGCCAGTGTaagaggaagatcgt (SEQ ID NO: 59)cctcgacTGTCGCTGCTCCGATATGCGTC 3′ 5′ GAGCAGCGACAgtcgaggacgatcttcctct (SEQID NO: 60) tACACTGGCACTGATCGCAACAGGGCTTAGA 3′i. BBI with a-Membrane Metalloprotease 2 (MMP2) Peptide in Loop I:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures,except the following two primers were used to introduce a peptidesequence that binds to MMP2 (denoted a-MMP2) to inhibit MMP2 function:5′ CAGTGCGCGTGTgccgccatgttcggccccg (SEQ ID NO: 61)ccTGTCGCTGCTCCGATATGCGTC 3′ 5′ GAGCAGCGACAggcggggccgaacatggcgg (SEQ IDNO: 62) cACACGCGCACTGATCGCAACAG 3′j. BBI with a-Membrane Metalloprotease 12 (MMP12) Peptide in Loop I:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures,except the following two primers were used to introduce a peptidesequence that binds to MMP12 (denoted a-MMP12) to inhibit MMP12function: 5′ CAGTGCGCGTGTggcgccctcggcctcttcg (SEQ ID NO: 63)gcTGTCGCTGCTCCGATATGCGTC 3′ 5′ GAGCAGCGACAgccgaagaggccgagggcgc (SEQ IDNO: 64) cACACGCGCACTGATCGCAACAG 3′k. BBI with Cotton Binding Peptide 2314 in Loop I:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures,except the following two primers were used to introduce a peptidesequence that binds to cotton: 5′ GTTGCGATCAGTGCGCGTGTgagcccctgat (SEQID NO: 65) ccaccagcgcTGTCGCTGCT 3′ 5′ CGCATATCGGAGCAGCGACAgcgctggtgga(SEQ ID NO: 66) tcaggggctcACACGCGCAC 3′l. BBI with Cotton Binding Peptide 2317 in Loop I:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures,except the following two primers were used to introduce a peptidesequence that binds to cotton: 5′ GTTGCGATCAGTGCGCGTGTagcgccttccg (SEQID NO: 67) cggccccaccTGTCGCTGCT3′ 5′ CGCATATCGGAGCAGCGACAggtggggccgc(SEQ ID NO: 68) ggaaggcgctACACGCGCAC 3′m. BBI with Compstatin Loop in Loop I:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures,except the following two primers were used to introduce the compstatinpeptide sequence: 5′ GTTGCGATCAGTGCGCGTGTgttgttcagga (SEQ ID NO: 69)ctggggccaccaccgcTGTCGCTGCT 5′ CGCATATCGGAGCAGCGACAgcggtggtggc (SEQ IDNO: 70) cccagtcctgaacaacACACGCGCAC

In this case, the 7 amino acids from the BBI Trypsin binding loop wasreplaced by 9 amino acids from compstatin binding loops.

n. BBI with Compstatin Loop in Loop II:

For plasmid construction, obtaining fungal transformants and assayingfungal transformant in shake flasks, we following same procedures,except the following two primers were used to introduce the compstatinpeptide sequence: 5′ CATGCAAGAGCTGTATCTGCgttgttcagga (SEQ ID NO: 71)ctggggccaccaccgcTGTTTCTGCG 5′ GTGATGTCGACGCAGAAACAgcggtggtggc (SEQ IDNO: 72) cccagtcctgaacaacGCAGATACAG

In this case, the 7 amino acids from the BBI Trypsin binding loop wasreplaced by 9 amino acids from compstatin binding loops.

Example 5 Expression of the Bowman-Birk Inhibitor and its Variants inTrichoderma reesei

Following procedures described in Example 2 above, the BBI-encoding DNAwas optimized and used for this Example. Two primers were designed toamplify the DNA fragment using plasmid pSLGAMpR2-BBI or pSLGAMpR2-BBIwith a-VEGF (CK37281) peptide in loop I and II as templates:

-   5′ GGA CTA GTA AGC GCG ACG ATG AGA GCT CT 3′ (SEQ ID NO:73)-   5′ AAG GCG CGC CTA GTT CTC CTT ATC GTC CT 3′ (SEQ ID NO:74)

A third primer was also used to create a PCR fragment which containsthree glycine residues at the N-terminal of the BBI protein when used inconjunction with primer #2 (SEQ ID NO:74) above. 5′ GGA CTA GTA AGC GCGGCG GTG GCG (SEQ ID NO: 75) ACG ATG AGA GCT CT 3′

Following the same procedures described in Example 2 above, theBBI-encoding DNA was optimized and used for this Example, the PCRfragment was cut with restriction enzyme SpeI and AscI and ligated tothe Trichoderma expression plasmid, pTrex4 (FIG. 8) which is a modifiedversion of pTREX2 (see FIG. 9), which in turn is a modified version ofpTEX, see PCT Publication No. WO 96/23928 for a complete description ofthe preparation of the pTEX vector, herein incorporated by reference,which contains a CBHI promoter and terminator for gene expression and aTrichoderma pyr4 gene as a selection marker for transformants, to createan expression plasmid. In the pTrex4 plasmid, the BBI gene was fused tothe C-terminus of the CBH I core and linker from T. reesei. The amdSgene from A. nidulans was used as the selection marker during fungaltransformation. The expression plasmid was transformed into Trichodermareesei. Stable transformants were isolated on Trichoderma minimal plateswith acetamide as the nitrogen source. The transformants were grown onthe amd minus plate which contains 1 ml/l 1000×salts, 20 g/l Noble Agar,1.68 g/l CsCl, 20 g/l Glucose, 15 g/l KH2PO4, 0.6 g/l MgSO4*7H2O, 0.6g/l CaCl2*2H2O and 0.6 g/l Acetamide. The final pH was adjusted to 4.5.The 1000× salts contains 5 g/l FeSO4, 1.6 g/l MnSO4, 1.4 g/l ZnSO4 and 1g/l CoCl2. It was filter sterilized. After three days incubation at 28°C., the transformants were transferred to the fresh amd minus plates andgrown for another three days at 28° C.

The transformants were then inoculated into T. reesei proflo medium (50ml for each transformant) in 250-ml shake flasks. T. reesei proflomedium contains 30 g/l Alpha-lactose, 6.5 g/l (NH4)2SO4, 2 g/l KH2PO4,0.3 g/l MgSO4*7H₂O, 0.2 g/l CaCl2, 1 ml/l 1000×TRI Trace Salts, 2m/l/10% Tween 80, 22.5 g/l Proflo and 0.72 g/l CaCO3. The 1000× TR1Trace Salts contains 5 g/l FeSO4*7H2O, 1.6 g/l MnSO4*H₂O and 1.4 g/lZnSO4*7H₂O. After growing at 30° C. for 2 days, 4 ml of culture wastransferred into defined medium which contains 5 g/l (NH4)2SO4, 33 g/lPIPPS buffer, 9 g/l CASAMINO ACIDS, 4.5 g/l KH2PO4, 1 g/l CACL2, 1 g/lMgSO4*7H₂O, 5 ml/l MAZU and 2.5 m/l/400× T. reesei TRACE. Its pH wasadjusted to 5.5 and 40 ml/1 40% lactose was added after sterilization.The 400× T. reesei TRACE contains 175 g/l Citric Acid (anhydrous), 200g/l FeSO4*7H₂O, 16 g/l ZnSO4*7H₂O, 3.2 g/l CuSO4*5H2O, 1.4 g/l MnSO4*H₂Oand 0.8 g/l H₃BO3 (Boric Acid).

About 40 transformants were generated on the plates and 20 were assayedin shake flasks. The supernatant of the culture was used for SDS-PAGEanalysis and assayed for trypsin or chymotrypsin inhibitory activity.Western blot also showed the presence of both fusion (CbhI-BBI) and BBIalone.

Example 6 Co-Expression of the Bowman-Birk Inhibitor and SecretoryChaperones in Aspergillus

The following example details how secretion can be enhanced. STI proteincontains two disulfide bonds and BBI contains 7 disulfide bonds in theirtertiary structures and these disulfide bonds are important for theirfunction. It is known that folding of protein with disulfide bondsrequire Protein Disulfide Isomerase (PDI) or other chaprones in ER.

Enhancement of STI or BBI expression was investigated byco-transformation of two plasmids or by sequential transformation of twoplasmids, one contains STI or BBI expression cassette and the other onecontains the PDI genes or chaperone genes. First, we co-transformplasmid pSLGAMpR2-BBI without 3G and histag (Q109) with plasmid Q51which contains 4.6 kb genomic DNA covering region of the pdiA gene fromAspergillus niger in vector pUC219 into same strain (dgr46ΔGAP:pyr2).Fifty-one transformants were obtained and forty-seven transformants werescreened in shake flasks. Transformant #14 was selected because itproduced the highest amount of BBI protein based on SDS gel data. Theexpression level of BBI protein is higher in the co-transformed stainthan the strain containing only plasmid pSLGAMpR2-BBI without 3G andhistag (Q109). FIG. 7 illustrates the enhanced BBI expression. Thisstrain was also spore purified and tested again in shake flask.

Following procedures described above, we also decide to co-transformplasmid pSLGAMpR2-BBI without histag (Q108) with plasmid Q51 containingpdiA gene (same as above) into same strain (dgr246ΔGAP:pyr2).Thirty-four transformants were screened in shake flasks. Onetransformant was selected for its ability to produce BBI protein at thehighest level based on the SDS gel date. The expression level of BBIprotein is higher than the strain containing only plasmid pSLGAMpR2-BBIwithout histag (Q108).

Following procedures described above, we also decide to co-transformplasmid pSLGAMpR2-BBI without 3G and histag (Q109) with plasmid Q124which contains 1623 bp genomic DNA covering region of the prpA gene fromAspergillus niger in vector pUC219 into same strain (dgr246ΔGAP:pyr2).Twenty-eight transformants were screened in shake flasks. Onetransformant was selected for its ability to produced the highest amountof BBI protein based on the SDS gel date. The expression level of BBIprotein is higher in the co-transformed stain than the strain containingonly plasmid pSLGAMpR2-BBI without 3G and histag (Q109). FIG. 7illustrates the enhancement (lane 15 vs lane 3). This strain was sporepurified and tested again in shake flask.

Example 7 Recombinant Protease Inhibitor Variants Retain Activity

STI, BBI and variants thereof produced using the methods described abovewere tested for activity, e.g., inhibition of protease activity.

a. Protease Inhibition

950 μl of Tris-buffered saline+0.02% Tween 20 is combined with 20 μlprotease (100 μg/ml in 1 mM HCl (bovine trypsin or chymotrypsin)) and 20μl sample. The solution is mixed and incubated for 30 min. at roomtemperature. 10 μl substrate (for trypsin:succinyl-ala-ala-pro-arg-paranitroanilide, 10 mg/ml in DMSO; forchymotrypsin: succinyl ala-ala-pro-phe-paranitroanilide, 10 mg/ml inDMSO) is added and the solution mixed. Absorbance is monitored at 405 nmand the rate determined (A₄₀₅/min). The fraction of protease activityinhibited is determined by comparison with a control sample blank andcalculated according to the following equation:$\left( \frac{A_{405}/{\min({sample})}}{A_{405}/{\min({blank})}} \right)*100\quad{\quad{{{\mu g}\text{/}\quad{{ml}({protease})}*\left( \frac{{MW}{inhibitor}}{{MW}{protease}} \right)} = {\lbrack{inhibitor}\rbrack\quad{\mu g}\text{/}{ml}}}}$b. Inhibition of HUVEC Proliferation by aVEGF Peptides.

HUVE cells (Cambrex, East Rutherford, N.J.) were passaged 1-5 times andmaintained according to manufacturers instructions. HUVEC growth wasstimulated by 0.03 to 20 ng/ml VEGF with the highest proliferation at 10ng/ml VEGF₁₆₅ (R&D systems); this concentration was used in subsequentexperiments. A series of a-VEGF peptides (see Example 4) from 0.00052 μMto 25 μM and an anti-VEGF MAb control (R&D Systems) were mixed with 10ng/mL VEGF prior to addition to HUVECs seeded in triplicate in 96-wellplates. Cell proliferation was measured by ³H-thymidine incorporation.Significant inhibition was observed (data not shown).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method for producing a protease inhibitor in a filamentous fungalcell comprising, a) introducing a DNA construct into a filamentousfungal cell, wherein said DNA construct comprises a promoter showingtranscription activity in the filamentous fungal cell and which isoperably linked to a heterologous DNA sequence encoding a proteaseinhibitor derived from a Soybean Trypsin Inhibitor (STI), a Bowman-BirkInhibitor (BBI) or variants thereof b) culturing the filamentous fungalcell under suitable culture conditions to allow expression of theheterologous DNA sequence, and c) producing the protease inhibitor. 2.The method according to claim 1 further comprising recovering theprotease inhibitor.
 3. The method according to claim 1, wherein thefilamentous fungal cell is selected from an Aspergillus strain, aPenicillium strain, a Fusarium strain or a Trichoderma strain.
 4. Themethod according to claim 3, wherein the Trichoderma strain is T.reesei.
 5. The method according to claim 3, wherein the Aspergillusstrain is A. niger, A. nidulans, A. awamori or A. oryzea.
 6. The methodaccording to claim 1, wherein the protease inhibitor is selected fromthe group consisting of a) a protease inhibitor having at least 90%sequence identity with SEQ ID NO: 7, b) a protease inhibitor having atleast 90% sequence identity with SEQ ID NO: 7 wherein residues 15-21and/or 42-47 have been replaced with a variant sequence, c) a proteaseinhibitor having at least 90% sequence identity with SEQ ID NO: 12, andd) a protease inhibitor having at least 90% sequence identity with SEQID NO: 12, wherein residues 39-85 and/or 137-144 have been replaced witha variant sequence.
 7. The method according to claim 6, wherein theprotease inhibitor has at least 90% sequence identity with SEQ ID NO: 7wherein residues 15-21 and/or 42-47 have been replaced with a variantsequence.
 8. The method according to claim 7, wherein the proteaseinhibitor is selected from the group consisting of SEQ ID NOs. 7 and15-29.
 9. The method according to claim 1, wherein the DNA sequenceencoding the protease inhibitor includes codons that have been optimizedfor expression in the filamentous fungal cell.
 10. The method accordingto claim 1 further comprising introducing a second nucleic acid sequenceencoding a chaperone into the filamentous fungal cell.
 11. The methodaccording to claim 10, wherein the chaperone is pdiA or prpA.
 12. Themethod according to claim 1, wherein the protease inhibitor is expressedas a fusion protein.
 13. The method according to claim 12, wherein thefusion protein includes a glucoamylase signal sequence, a glucoamylasecatalytic domain, a cleavage site, and the protease inhibitor.
 14. Themethod according to claim 12, wherein the fusion protein is processed bya protease to liberate the protease inhibitor.
 15. A protease inhibitorcomposition comprising the protease inhibitor produced according to themethod of claim
 1. 16. A method of inhibiting the proteolytic activityof a target protein comprising contacting the target protein with theprotease inhibitor composition of claim 15 and binding the targetprotein wherein the proteolytic activity of said target protein isinhibited.
 17. An isolated polynucleotide encoding a protease inhibitorselected from the group consisting of polypeptide sequences set forth inSEQ ID NOs: 15-29.
 18. An altered wild-type polynucleotide encoding aSoybean Trypsin Inhibitor or Bowman Birk Inhibitor comprising codonswhich have been optimized for expression in a filamentous fungal cell.19. An expression vector comprising (a) a polynucleotide sequenceencoding a protease inhibitor having at least 90% sequence identity withSEQ ID NO: 12, wherein residues 39-85 and/or 137-144 have been replacedwith a variant sequence or (b) a protease inhibitor having at least 90%sequence identity with SEQ ID NO: 7, wherein residues 15-21 and/or 42-47have been replaced with a variant sequence.
 20. The expression vector ofclaim 19, further comprising from the 5′ terminus to the 3′ terminus, afirst nucleic acid sequence encoding a signal peptide functional as asecretory sequence in a filamentous fungus, a second nucleic acidsequence encoding a secreted polypetide or functional portion thereof, athird nucleic acid sequence encoding a cleavable linker, and the DNAsequence which encodes the protease inhibitor.
 21. A host celltransformed with the vector of claim
 19. 22. The host cell of claim 21,wherein said host cell is a Trichoderma cell.
 23. The host cell of claim21, wherein said host cell is an Aspergillus cell.
 24. A method forenhancing the expression of a protease inhibitor in a filamentous fungalcell comprising a) transforming a filamentous fungal cell with a DNAconstruct which comprises a promoter showing transcription activity inthe filamentous fungal cell and which is operably linked to aheterologous DNA sequence encoding a protease inhibitor derived from aSoybean Trypsin Inhibitor (STI), a Bowman-Birk Inhibitor (BBI), orvariants thereof, b) transforming the filamentous fungal cell with apolynucleotide sequence containing a chaperone gene, and c) culturingthe filamentous fungal cell under suitable culture conditions to allowexpression and secretion of the heterologous DNA sequence encoding theprotease inhibitor wherein expression of the protease inhibitor isenhanced compared to a corresponding filamentous fungal cell transformedonly in accordance with step a).
 25. The method according to claim 24,wherein the transformation step a) and the transformation step b) is aco-transformation.
 26. The method according to claim 24, wherein thetransformation step a) and transformation step b) is a sequentialtransformation.
 27. The method according to claim 24, wherein thefilamentous fungal cell is an Aspergillus cell or a Trichoderma cell.